Treatment of heart disease by inhibition of the action of ribosomal s6 kinase 3 (rsk3)

ABSTRACT

The present invention provides a method of protecting the heart from damage, by administering to a patient at risk of such damage, a pharmaceutically effective amount of a composition which inhibits the interaction of RSK3 and mAKAPβ, or the expression or activity of one or both of those molecules. This composition may be in the form of a peptide that specifically inhibits mAKAPβ binding to RSK3 or in the form of an siRNA construct which inhibits the expression of RSK3.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of and claims priority to pendingU.S. patent application Ser. No. 14/213,583, filed on Mar. 14, 2014,which claims the benefit of U.S. Provisional Application No. 61/798,268,filed Mar. 15, 2013, each of which are hereby incorporated by referencein their entireties into the present application.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with Government support under contract RO1 HL075398 awarded by the National Institutes of Health. The Government hascertain rights in this invention.

BACKGROUND OF THE INVENTION

The heart is capable of undergoing hypertrophic growth in response to avariety of stimuli.

Hypertrophic growth may occur as the result of physical training such asrunning or swimming. However, it also occurs as the result of injury orin many forms of heart disease. Hypertrophy is the primary mechanism bywhich the heart reduces stress on the ventricular wall. When the growthis not accompanied by a concomitant increase in chamber size, this iscalled concentric hypertrophy. Hypertrophy occurs as the result of anincrease in protein synthesis and in the size and organization ofsarcomeres within individual myocytes. For a more thorough review ofcardiac remodeling and hypertrophy, see Kehat (2010) and Hill (2008),each herein incorporated by reference in their entirety. The prevailingview is that cardiac hypertrophy plays a major role in heart failure.Traditional routes of treating heart failure include afterloadreduction, blockage of beta-adrenergic receptors (β-ARs) and use ofmechanical support devices in afflicted patients. However, the art is inneed of additional mechanisms of preventing or treating cardiachypertrophy.

AKAPs and Cardiac Hypertrophy

Ventricular myocyte hypertrophy is the primary compensatory mechanismwhereby the myocardium reduces ventricular wall tension when submittedto stress because of myocardial infarction, hypertension, and congenitalheart disease or neurohumoral activation. It is associated with anonmitotic growth of cardiomyocytes, increased myofibrillarorganization, and upregulation of specific subsets of “fetal” genes thatare normally expressed during embryonic life (Frey 2004, Hill 2008). Theconcomitant aberrant cardiac contractility, Ca²⁺ handling, andmyocardial energetics are associated with maladaptive changes thatinclude interstitial fibrosis and cardiomyocyte death and increase therisk of developing heart failure and malignant arrhythmia (Cappola 2008,Hill 2008). Increased in prevalence by risk factors such as smoking andobesity, heart failure is a syndrome that affects about six millionAmericans and has an annual incidence of 1% of senior citizens (Roger2011). Since the five-year survival rate after diagnosis is still verypoor (lower than 50%), many efforts have been made during the last yearsto define the molecular mechanisms involved in this pathologicalprocess.

Cardiac hypertrophy can be induced by a variety of neuro-humoral,paracrine, and autocrine stimuli, which activate several receptorfamilies including G protein-coupled receptors, cytokine receptors, andgrowth factor tyrosine kinase receptors (Brown 2006, Frey 2004). In thiscontext, it is becoming increasingly clear that AKAPs can assemblemultiprotein complexes that integrate hypertrophic pathways emanatingfrom these receptors. In particular, recent studies have now identifiedanchoring proteins including mAKAP and AKAP-Lbc and D-AKAP1 that play acentral role in organizing and modulating hypertrophic pathwaysactivated by stress signals.

mAKAP.

In cardiomyocytes, mAKAPβ is localized to the nuclear envelope throughan interaction with nesprin-1α (Pare 2007). mAKAPβ assembles a largesignaling complex that integrates hypertrophic signals initiated byα₁-adrenergic receptors (α₁-ARs) and β-ARs, endothelin-1 receptors, andgp130/leukemia inhibitor factor receptors (FIG. 46A) (Dodge-Kafka 2005,Pare 2005). Over the last few years, the molecular mechanisms as well asthe signaling pathways whereby mAKAPβ mediates cardiomyocyte hypertrophyhave been extensively investigated. It is now demonstrated that mAKAPβcan recruit the phosphatase calcineurin A|3 (CaNAβ) as well as thehypertrophic transcription factor nuclear factor of activated T cells c3(NFATc3) (Li 2010). In response to adrenergic receptor activation,anchored CaNAβ dephosphorylates and activates NFATc3, which promotes thetranscription of hypertrophic genes (FIG. 46A) (Li 2010). The molecularmechanisms controlling the activation of the pool of CaNAβ bound to themAKAPβ complex are currently not completely understood but seem torequire mobilization of local Ca²⁺ stores. In this context, it has beenshown that mAKAP favors PKA-induced phosphorylation of RyR2 (Kapiloff2001), which, through the modulation of perinuclear Ca²⁺ release, couldactivate CaNAβ (FIG. 46A). In line with this hypothesis, the deletion ofthe PKA anchoring domain from mAKAPβ has been shown to suppress themAKAP-mediated hypertrophic response (Pare 2005). On the other hand,recent findings indicate that mAKAPβ also binds phospholipase Cε(PLCε)and that disruption of endogenous mAKAPβ-PLCεcomplexes in rat neonatalventricular myocytes inhibits endothelin 1-induced hypertrophy (Zhang2011). This suggests that the anchoring of PLCε to the nuclear envelopeby mAKAPβ controls hypertrophic remodeling. Therefore, it is alsoplausible that at the nuclear envelope, PLCε might promote thegeneration of inositol 1,4,5-trisphosphate, which through themobilization of local Ca²⁺ stores, might promote the activation of CaNAβand NFATc3 bound to mAKAPβ (FIG. 46A).

In cardiomyocytes, the dynamics of PKA activation within the mAKAPcomplex are tightly regulated by AC5 (Kapiloff 2009) and the PDE4D3(Dodge-Kafka 2005, Dodge 2001) that are directly bound to the anchoringprotein. The mAKAP-bound AC5 and upstream β-AR may be localized withintransverse tubules adjacent to the nuclear envelope (Escobar 2011). Inresponse to elevated cAMP levels, mAKAP-bound PKA phosphorylates bothAC5 and PDE4D3 (Dodge-Kafka 2005, Dodge 2001, Kapiloff 2009). Thisinduces AC5 deactivation and PDE4D3 activation, which locally decreasescAMP concentration and induces deactivation of anchored PKA (FIG. 46A).Dephosphorylation of PDE4D3 is mediated by the phosphatase PP2A that isalso associated with mAKAPβ (FIG. 46A) (Dodge-Kafka 2010). Collectively,these findings suggest that the mAKAP complex generates cyclic pulses ofPKA activity, a hypothesis that was supported experimentally by livecell imaging studies (Dodge-Kafka 2005).

AKAPs and Hypoxia

Myocardial oxygen levels need to be maintained within narrow levels tosustain cardiac function. During ischemic insult, in response toconditions of reduced oxygen supply (termed hypoxia), cardiomyocytesmobilize hypoxia-inducible factor 1α (HIF-1α), a transcription factorthat promotes a wide range of cellular responses necessary to adapt toreduced oxygen (Semenza 2007). Transcriptional responses activated byHIF-1α control cell survival, oxygen transport, energy metabolism, andangiogenesis (Semenza 2007). Under normoxic conditions, HIF-1α ishydroxylated on two specific proline residues by the prolyl hydroxylasedomain proteins (PHDs) and subsequently recognized and ubiquitinated bythe von Hippel-Lindau protein (Jaakkola 2001, Maxwell 1999).Ubiquitinated HIF-la is targeted to the proteasome for degradation. Onthe other hand, when oxygen concentration falls, the enzymatic activityof PHD proteins is inhibited. Moreover, PHD proteins are ubiquitinatedby an E3 ligase named “seven in absentia homolog 2 (Siah2)” and targetedfor proteasomal degradation (Nakayama 2004). This inhibits HIF-1αdegradation and allows the protein to accumulate in the nucleus where itpromotes gene transcription required for the adaptive response tohypoxia. In line with this finding, the delivery of exogenous HIF-1αimproves heart function after myocardial infarction (Shyu 2002), whereascardiac overexpression of HIF-1α reduces infarct size and favors theformation of capillaries (Kido 2005).

Recent findings indicate that mAKAP assembles a signaling complexcontaining HIF-1α, PHD, von Hippel-Lindau protein, and Siah2 (Wong2008). This positions HIF-1α in proximity of its upstream regulators aswell as to its site of action inside the nucleus. In this configuration,under normoxic conditions, negative regulators associated with the mAKAPcomplex favor HIF-1α degradation (Wong 2008). On the other hand, duringhypoxia, the activation of Siah2 within the mAKAP complex promotesHIF-1α stabilization, allowing the transcription factor to inducetranscription (Wong 2008). Therefore, mAKAP assembles a macromolecularcomplex that can favor degradation or stabilization of HIF-1α incardiomyocytes in response to variations of oxygen concentrations. Inthis context, mAKAP could play an important role in cardiomyocyteprotection during cardiac ischemia, when coronary blood flow is reducedor interrupted. By coordinating the molecular pathways that controlHIF-1α stabilization in cardiomyocytes, mAKAP might favorHIF-1α-mediated transcriptional responses, controlling the induction ofglycolysis (which maximizes ATP production under hypoxic conditions),the efficiency of mitochondrial respiration, and cell survival duringischemia (Semenza 2009).

Myofibrillar assembly driving nonmitotic growth of the cardiac myocyteis the major response of the heart to increased workload (Kehay 2010).Although myocyte hypertrophy per se may be compensatory, in diseasessuch as hypertension and myocardial infarction, activation of thehypertrophic signaling network also results in altered gene expression(“fetal”) and increased cellular apoptosis and interstitial fibrosis,such that left ventricular hypertrophy is a major risk factor for heartfailure. Current therapy for pathologic hypertrophy is generally limitedto the broad downregulation of signaling pathways through the inhibitionof upstream cell membrane receptors and ion channels (McKinsey 2007).Novel drug targets may be revealed through the identification ofsignaling enzymes that regulate distinct pathways within thehypertrophic signaling network because of isoform specificity orassociation with unique multimolecular signaling complexes.

p90 ribosomal S6 kinases (RSK) are pleiotropic extracellularsignal-regulated kinase (ERK) effectors with activity that is increasedin myocytes by most hypertrophic stimuli (Anjum 2008, Sadoshima 2005,Kodama 2000). In addition, increased RSK activity has been detected inexplanted hearts from patients with end-stage dilated cardiomyopathy(Takeishi 2002). There are 4 mammalian RSK family members that areubiquitously expressed and that overlap in substrate specificity (Anjum2008). RSKs are unusual in that they contain 2 catalytic domains,N-terminal kinase domain and C-terminal kinase domain (FIG. 4A, Anjum2008). The N-terminal kinase domain phosphorylates RSK substrates and isactivated by sequential phosphorylation of the C-terminal kinase domainand N-terminal kinase domain by ERK (ERK1, ERK2, or ERK5) and3′-phosphoinositide-dependent kinase 1 (PDK1), respectively (Anjum2008).

By binding scaffold proteins, RSKs may be differentially localizedwithin subcellular compartments, conferring isoform-specific signalingbound to the scaffold protein muscle A-kinase anchoring protein (mAKAP)(Michael 2005). PDK1 activation of RSK was enhanced by co-expressionwith the mAKAP scaffold in a recombinant system. In cardiac myocytes,mAKAPβ (the alternatively spliced form expressed in muscle cells)organizes signalosomes that transduce cAMP, mitogen-activated proteinkinase, Ca²⁺, and hypoxic signaling by binding a diverse set of enzymes,ion channels, and transcription factors (Kritzer 2012).

SUMMARY OF THE INVENTION

The following brief summary is not intended to include all features andaspects of the present invention, nor does it imply that the inventionmust include all features and aspects discussed in this summary.

The present inventors have discovered methods of treating cardiacpathological processes by inhibiting the signaling properties ofindividual mAKAP signaling complexes using drugs that target uniqueprotein-protein interactions. Such a therapeutic strategy offers anadvantage over classical therapeutic approaches because it allows theselective inhibition of defined cellular responses.

In particular, the present inventors have found that disruptingmAKAP-mediated protein-protein interactions can be used to inhibit theability of mAKAP to coordinate the activation of enzymes that play acentral role in activating key transcription factors that initiate theremodeling process leading to cardiac hypertrophy.

Specifically, the inventors have discovered that inhibiting the bindinginteraction between type 3 ribosomal S6 kinase (RSK3) and mAKAPβ canprotect the heart from damage caused by various physical stresses, forexample pressure overload and prolonged exposure to high levels ofcatecholamines.

Thus, the present invention comprises, in certain aspects a method forprotecting the heart from damage, by administering to a patient at riskof such damage, a pharmaceutically effective amount of a compositionwhich inhibits the interaction of RSK3 and mAKAPβ.

The invention also relates to a method of treating heart disease, byadministering to a patient a pharmaceutically effective amount of acomposition which inhibits the interaction of RSK3 and mAKAPβ.

The invention also relates to compositions which inhibit the interactionof RSK3 and mAKAPβ.

In still other embodiments, the inhibitors include any molecule thatinhibits the expression or activity of of RSK3 and mAKAPβ.

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Model for RSK3 signaling. MAP-Kinase signaling induced byα₁-adrenergic receptor (α₁-AR) stimulation and potentially otherupstream signals activates anchored RSK3 in conjunction with PDK1 atanchored sites, including at perinuclear mAKAPβ signalosomes. Targetsfor RSK3 may include cytosolic and nuclear proteins, especially thoseinvolved in the regulation of hypertrophic gene expression.

FIG. 2. Shows the amino acid sequence of RSK3 (SEQ ID NO: 1).

FIG. 3. Shows the amino acid sequence of mAKAPβ (SEQ ID NO: 2).

FIG. 4. The unique p90 ribosomal S6 kinase type 3 (RSK3) N-terminaldomain binds muscle A-kinase anchoring protein (mAKAP). A, p90 ribosomalS6 kinases (RSKs) contain 2 catalytic domains, N-terminal kinase domain(NTKD) and C-terminal kinase terminal (CTKD).³ Only RSK3 has anN-terminal nuclear localization signal (NLS). This NLS is within thesame unique region of RSK3 that binds mAKAPβ. In inactive RSK3, the CTKDbinds the autoinhibitory domain (AID) α-helix. Prebound to the D-domain,when activated, extracellular signal-regulated kinase (ERK)phosphorylates RSK3 residues including the CTKD activation loop (T⁵⁷⁰).The CTKD then autophosphorylates S³⁷⁷, permittingphosphoinositide-dependent kinase 1 (PDK1) binding and phosphorylationof the NTKD activation loop (S²¹⁸). The NTKD phosphorylates RSKsubstrates. RSKs 1 to 4 were aligned and sequence similarity wascalculated using Vector NTI AlignX (Invitrogen). RSK3 fragments used formapping are indicated. B, Rat neonatal myocyte extract (lane 1, 0.2%total extract) was immunoprecipitated with preimmune (lane 2) oranti-VO56 mAKAP (lane 3) sera and detected using a pan-RSK antibody(C-20; see FIG. 21 legend). C, HA-tagged kinases and myc-tagged mAKAPawere expressed by cotransfection of HEK293 cells andcoimmunoprecipitated with anti-HA antibody. D, HA-tagged RSK3 fragmentsand myc-tagged mAKAPβ were expressed by cotransfection of COS-7 cellsand coimmunoprecipitated with anti-HA antibody. n≧3 for each panel.

FIG. 5. p90 ribosomal S6 kinase type 3 (RSK3) signaling is important forneonatal rat ventricular myocyte hypertrophy. A, Neonatal myocytes wereinfected with Adeno-HA-RSK3 for 1 day in maintenance media before beingserum-starved for 2 days and then treated for 1 hour with 10 μmol/Lphenylephrine (PE), 10 μmol/L BIX02189, or 0.1 μmol/L PD0325901. Wholecell extracts were used for immunoblotting (n=3). B-F, Myocytes weretransfected with control or RSK3 siRNA oligonucleotides and cultured for2 days±10% fetal bovine serum (FBS), 10 μmol/L PE, or 1000 U/mL leukemiainhibitory factor (LIF). B, RSK3 reverse-transcriptase polymerase chainreaction (RT-PCR; top) and Western blot for RSK3 immunoprecipitated withN-16 antibody (bottom) using PE-treated myocytes. C, Results obtained byTUNEL staining (n=3). D, Immunocytochemistry for α-actinin (green),atrial natriuretic factor (ANF; red), and Hoechst (blue); bar=20 μm.Separate ANF and Hoechst channels are provided in FIG. 23. E,Cross-section area of myocytes (n=4-8). F, Fraction of myocytesexpressing ANF (n=4-5). *P values comparing samples treated with thesame hypertrophic agonist. ^(†)P values compared with no hypertrophicagonist control.

FIG. 6. Inhibition of neonatal rat ventricular myocyte hypertrophy withp90 ribosomal S6 kinase (RSK) inhibitor. Myocytes were cultured for 2days±10 μmol/L phenylephrine (PE), 1000 U/mL leukemia inhibitory factor(LIF), 10% fetal bovine serum (FBS), or 10 μmol/L BI-D1870 or 1% DMSOcarrier. A, Immunocytochemistry for α-actinin (green), atrialnatriuretic factor (ANF; red), and Hoechst (blue); bar=20 μm. SeparateANF and Hoechst channels are provided in FIG. 25. B, Cross-section areaof myocytes (n=3-7). C, Fraction of myocytes expressing ANF (n=3). *Pvalues compared with DMSO. ^(†)P values compared with no agonistcontrol.

FIG. 7. The p90 ribosomal S6 kinase type 3 (RSK3) binding site withinmuscle A-kinase anchoring protein (mAKAP). A, mAKAP domain structure.Direct binding partners whose sites have been finely mapped in mAKAPβare shown (Li 2010, Pare 2005, Zhang 2011, Dodge-kafka 2010, Kapiloff2009, Kapiloff 1999, Marx 2001). mAKAPβ starts at residue 245 of mAKAPa.All fragments are numbered per mAKAPa. The grey bar indicates the RSK3binding site. B-F, Specific tag antibodies were used toimmunoprecipitate myc-tagged mAKAPβ and HA-tagged p90 ribosomal S6kinase (RSK) proteins (see FIG. 4A) co-expressed in either HEK293 (B) orCOS-7 (C-F) cells (n=3) for all panels. G, Surface plasmon resonanceusing purified bacterially expressed proteins. Unphosphorylated (left),extracellular-regulated signaling kinase (ERK)-phosphorylated (middle),and ERK and phosphoinositide-dependent kinase 1 (PDK1)-phosphorylated(right) His-RSK3 (12.5-200 nmol/L in perfusate) were bound to His-mAKAP1286 to 1833 (solid state). Each curve was repeated 3 times usingdifferent protein preparations.

FIG. 8. p90 ribosomal S6 kinase type 3 (RSK3) anchoring is important forneonatal rat ventricular myocyte hypertrophy. A, Muscle A-kinaseanchoring protein beta (mAKAPβ) complexes were immunoprecipitated usingFL100 mAKAP antiserum from phenylephrine (PE)-treated,adenovirus-infected myocytes expressing myc-green fluorescent protein(GFP) or myc-GFP-mAKAP RSK binding domain (RBD) fusion protein (GFP-RBD;mAKAP 1694-1833) and detected with the pan-RSK 1F6 and mAKAP 211antibodies. B, Transfected myocytes expressing GFP or GFP-RBD (green)were stained with α-actinin (blue) and atrial natriuretic factor (ANF;red) antibodies. Bar=20 μm. C, Cross-section area of myocytes (n=5). D,Fraction of myocytes expressing ANF (n=3). *P values comparing toGFP-expressing samples. ^(†)P values compared with no agonist control.

FIG. 9. p90 ribosomal S6 kinase type 3 (RSK3) knockout attenuates theeffects of pressure overload in vivo. A, Hematoxylin and eosin-stainedtransverse sections. Bar=1 mm. B, Wheat-germ agglutinin-stainedsections. Bar=50 μm. C, Cross-section area of myocytes in tissuesections (n=4-9). D, Bright field images of acutely dissociated adultcardiac myocytes. Bar=50 μm. Width (E), length (F), and length/widthratio (G) of isolated myocytes (n=5-6). ^(†)P values compared withsham-operated mice of the same genotype; *P values compared withRSK3^(+/+) transverse aortic constriction (TAC) mice.

FIG. 10 shows echocardiographic data for RSK^(−/−) mice after transverseaortic constriction.

FIG. 11 shows gene expression for RSK^(−/−) mice after transverse aorticconstriction.

FIG. 12 shows breeding of RSK^(−/−) mice.

FIG. 13 shows echocardiographic data for RSK^(−/−) mice when unstressed.

FIG. 14 shows gravimetric data for RSK^(−/−) mice following TAC.

FIG. 15 shows echocardiographic data for RSK^(−/−) mice afterisoproterenol infusion.

FIG. 16 shows gravimetric data for RSK^(−/−) mice after isoproterenolinfusion.

FIG. 17 shows echocardiographic data for RSK^(−/−) mice after chronicexercise.

FIG. 18 shows gravimetric data for RSK^(−/−) mice after chronicexercise.

FIG. 19 shows sources of commercial antibodies used.

FIG. 20 shows sequences of oligonucleotides (SEQ ID NOs: 3-8).

FIG. 21 Specificity of RSK3 Antibodies. A. HA-tagged RSK1, RSK2, RSK3(in duplicate at different concentrations), RSK4, and MSK2 wereexpressed in COS-7 cells, and whole cell lysates were used for westernblots with the indicated antibodies. B. Alignment of RSK and MSK familymembers (RSK1: SEQ ID NOs: 9 and 10; RSK2: SEQ ID NOs: 11 and 12; RSK3:SEQ ID NOs: 13 and 14; RSK4: SEQ ID NOs: 15 and 16; MSK1: SEQ ID NOs: 17and 18; MSK1: SEQ ID NOs: 19 and 20). MSK1 and MSK2 are the only twoother mammalian protein kinases that like RSK have a NTKD and a CTKD.When affinity purified, the OR43 antibody is highly selective for RSK3.The inventors did not test the Cell Signaling rabbit anti-RSK3 antibody#9343, since it is apparently RSK3-specific as shown by data provided bythe manufacturer. The inventors also did not test the Santa Cruz goatanti-RSK3 antibody C-20 (sc-1431) used for immunoprecipitation of RSK3,since according to the manufacturer C-20 detects both RSK3 and to alesser extent RSK2. The Santa Cruz goat anti-RSK3 antibody N-16(sc-13378), also used for immunoprecipitation, is specific for RSK3since the antigen was a peptide within the unique RSK3 N-terminusaccording to the manufacturer and as evident from our experiments.However, the monoclonal mouse anti-RSK3 antibody 1F6 (also sold as M01)sold by a variety of companies also readily detects RSK2. As might beexpected from the conservation of the antigen sequence, the “RSK3”phospho-specific antibodies readily detected other RSK and MSK familymembers. Phosphorylation of S²¹⁸ was associated with a decreasedmobility of most of the enzymes in SDS-PAGE, consistent with themultiple phosphorylation events that would be expected prior to PDK1phosphorylation of that residue (Cf. FIGS. 22 and 5A). Note that therelatively increased detection of RSK4 with this antibody is consistentwith RSK4 being constitutive active in cells.² Similarly, the relativelyhigh signals for phospho-RSK3 are more likely attributable to anenhanced baseline ERK phosphorylation of RSK3 in cells than an increasedspecificity of this antibody for the RSK3 isoform.⁸ RSK3 is a minorityof the total RSK protein in myocytes (see FIG. 24C). As a result, signalderived from endogenous proteins in whole cell lysates using thesephospho-specific antibodies cannot be attributed solely to RSK3.

FIG. 22. RSK Activation in Neonatal Cardiac Myocytes. Neonatal ratventricular myocytes were treated for 1 hour with 10 μmol/L PE, 10% FBS,or 1000 U/mL LIF in the absence or presence of the MAPK inhibitors asindicated. Total RSK was detected using the non-specific monoclonal RSK3antibody 1F6 and the phospho-S²¹⁸ antibody. *p<0.05 relative to samecondition without inhibitor; ^(††)p<0.005 relative to control; n=2-7.Note that the graphs represent a compilation of the experimentspresented in A and B, and therefore, the PE bars are the same in bothgraphs. PD0325901 is selective for MEK1/2 at 0.1 μmol/L, but alsoinhibits MEK5 at 10 μmol/L.⁹ BIX02189 is specific for MEK5. SP600125 andSB103580 are selective for JNK and p38, respectively.

FIG. 23 shows grayscale images of FIG. 5D

FIG. 24. Active RSK3 induces neonatal rat ventricular myocytehypertrophy. Myocytes transfected with control or RSK3 siRNAoligonucleotides and infected with adenovirus to express GFP, HA-RSK3 orHA-RSK3 S²¹⁸A were cultured for 2 days±10 μmol/L PE. A.Immunocytochemistry for α-actinin (red) and Hoechst (blue); bar=20 μm.B. Cross-section area of myocytes. n=4-5. p<0.05: ^(†) compared to noagonist control; ‡, compared to control siRNA+GFP+PE; * compared tosimilarly treated GFP+RSK3 siRNA samples; ^(§) compared to similarlytreated HA-RSK3 WT+RSK3 siRNA samples. C. Whole extracts and RSK3immunoprecipitated with N-16 antibody were assayed by western blot. RSK3in the immunoprecipitates and RSK in whole cell extracts were detectedwith purified OR43 and mouse 1F6 antibodies, respectively. Red asterisksindicate detectable endogenous and HA-tagged RSK3 proteins. Note thatthe signal obtained with both the phospho-S²¹⁸ and OR43 RSK3 antibodieswere attenuated by the RSK3 siRNA and enhanced by HA-RSK3 expression. Nosignal was obtained for the HA-RSK3 S218A mutant with the phospho-S²¹⁸antibody. The level of expression of HA-RSK3 in non-treated cells wassimilar to the level of expression of endogenous RSK3 in PE-treatedcells. Importantly, neither expression of HA-tagged proteins, nor theuse of the siRNA, affected the signal obtained using the pan-RSKantibody for whole cell extracts.

FIG. 25 shows grayscale images of FIG. 6A.

FIG. 26 shows Inhibition of neonatal rat ventricular myocyte hypertrophywith the RSK active site inhibitor SL0101. Myocytes were cultured for 2days±10 μmol/L PE, 1000 U/mL LIF and/or 50 μmol/L SL0101 or 0.1% DMSOcarrier. A. Immunocytochemistry for-actinin (red) and Hoechst (blue);bar=20 μm. B. Cross-section area of myocytes. n=5. ^(†) compared to noagonist control; * compared to no SL0101.

FIG. 27. Design of RSK3 Knock-out Mouse. A. A PGKneo cassette wasinserted into Exon 2 interrupting translation at amino acid residue 67within the ATP-binding cassette of the RSK3 NTKD. Fidelity of homologousrecombination was confirmed using external probes for southern blottingand by PCR using the primers shown in black (sequence not shown). Priorto any experimentation, the PGKneo cassette was removed by matingtargeted mice to a global cre trangenic mouse. PCR primers (red) usedfor genotyping were RSK3Ex2+5′ and RSK3Ex2-3′ that produce 336 and 421bp fragments for wildtype and knock-out alleles, respectively. B.Example of mouse tail genotyping. C. Western blot of RSK3 proteinsdetected using OR43 RSK3 antibody following immunoprecipitation by N-16or C-20 antibodies. No RSK3 protein was detected in the knock-out mousehearts. Western blot of whole heart extracts with the mouse 1F6 antibody(left panel) showed that despite the loss of RSK3 protein, total RSKdetected in the knock-out hearts was the same as in wildtype.

FIG. 28. Echocardiography. Representative M-mode tracings forunstressed, sham-operated and TAC, saline and isoproterenol infused, andrested and swam mice. See FIGS. 10, 13, 15 and 16 for results.

FIG. 29. RSK3 is required for Isoproterenol-induced myocyte growth inwidth in vivo. A. Cross-section area of myocytes in tissue sections.n=3-5. Width (B), Length (C) and Length/Width Ratio (D) of acutelydissociated adult cardiac myocytes. n=5-6. ^(†)p-values comparing tountreated RSK3^(+/+) mice; ^(‡) p-values comparing to saline-infusedRSK3^(+/+) mice; * p-values comparing to Iso-infused RSK3^(+/+) mice.

FIG. 30. Phosphorylation of RSK3, ERK1/2, and known RSK substrates inmice hearts. 150 μg total heart extracts were analyzed by western blotfor the indicated proteins for 2 weeks TAC (A) and isoproterenolinfusion (B) cohorts. GSK-3βS⁹, myosin binding protein C (cMyBP-C) S²⁸²,eukaryotic elongation factor-2 kinase (eEF2K) S³⁶⁶, and cardiac troponinI (TnI) S^(23/24) are known RSK substrates. Mouse IF6 RSK antibody wasused to assay total RSK protein. No consistent changes in levels oftotal protein or phosphorylated proteins were detected.

FIG. 31 shows echocardiographic data for mice treated as in Example 2 at16 weeks of age.

FIG. 32. TM180 C57BL/6; FVB/N mice have a small heart phenotype. A. Toppanel: RSK3 protein was immunoprecipitated using N-16 antibody anddetected using OR43 antibody. Middle panel: Total RSK protein in heartextracts was detected using mouse anti-RSK antibody. Bottom panel:Ponceau staining for total heart protein shows that the major α-TMwildtype band is replaced by a lower TM180 band in transgenic hearts(Prabhakar 2001). n=3 B. Biventricular weight indexed to tibial length.C. Wheat germ agglutinin-strained heart sections. Bar=50 n=6 for eachcohort. D. Wet lung weight indexed to tibial length. *p-values comparedto WT cohort; ^(†)p-values compared to TM180 cohort. Cf FIG. 34 forpanels B and D.

FIG. 33. RSK3 is required for TM180 induced interstitial fibrosis. A.Trichrome staining of transverse sections. B. Higher magnification oftrichrome stained section. Bar=100 μm. C. Picrosirius red staining ofleft ventricular sections and fibrillar collagen content quantified bylinearly polarized light microscopy. Bar=200 μm. n=10-13;p(ANOVA)=0.002. D and E. Col8A1 (periostin) mRNA levels. n=3. Cf FIG.35. *p-values compared to WT cohort; ^(†)p-values compared to TM180cohort.

FIG. 34 shows gravimetric data for mice treated as in Example 2.

FIG. 35 shows gene expression data from the method of Example 2.

FIG. 36 shows electrocardiography from the mice treated as in Example 2.A. Representative M-mode images for 16 week old mice. See FIG. 31 forvalues. B. Left ventricular internal diameter in diastole (LVID; d) formice at the indicated ages by Mmode.

C. Fractional shortening (%) by M-Mode for mice at the indicated ages.D. Endocardial Percent Fractional Area Change (Endocardial FAC) byB-Mode for mice at the indicated ages. * p-values when compared to WT;^(†)p-values when compared to TM180. n=15-19 for each cohort. At allages, the TM180 mice had significantly smaller internal LVID; d andsignificantly higher fractional shortening and Endocardial FAC thanwildtype mice. At all ages, the TM180; RSK3−/− mice had mean values forthese parameters that were closer to wildtype than those for the TM180mice, albeit significance (p<0.05) between the TM180 and TM180; RSK3−/−mice cohorts was not reached for all of the age groups for eachparameter.

FIG. 37 shows mAKAPβ as a scaffold for signaling molecules important forcardiac stress responses.

FIG. 38 shows the mAKAPβ signalosome.

FIG. 39 shows a conditional mAKAP (AKAP6) knock-out mouse allele.

FIG. 40 shows that mAKAPβ is important in the cardiac myocyte for theresponse to pressure overload.

FIG. 41 shows AAV2/9 transduced expression of anchoring disruptorpeptides in vivo.

FIG. 42 shows results from a pilot study where mice were injectedintraperitoneally as neonates and subjected to 2 week transverse aorticconstriction (TAC) at 8 weeks of age.

FIG. 43 is the complete nucleotide (SEQ ID NO: 21) and amino acid (SEQID NO: 22) sequences of human RSK3. (Zhao et al. (1995) Mol. Cell. Biol.1995, 15(8):4353.)

FIG. 44. Shows the nucleotide sequence of RSK3 (SEQ ID NO: 23).

FIG. 45. Shows the nucleotide sequence of mAKAPβ (SEQ ID NO: 24).

FIG. 46. Regulation of cardiac hypertrophy by AKAP complexes. A: mAKAPassembles a multienzyme signaling complex at the outer nuclear membranecontaining AC5, PKA, PDE4D3, PP2A, RyR2, calcineurin Aβ (CaNAβ), nuclearfactor of activated T cells 3 (NFATc3), exchange protein activated bycAMP 1 (Epac1), and ERK5. Activation of AC5 by β-adrenergic stimulationgenerates cAMP, which in turn activates anchored PKA at submicromolarconcentrations. In a negative feedback loop, activated PKAphosphorylates PDE4D3, leading to its activation and increased cAMPdegradation, and AC5, leading to its inactivation and decreased cAMPsynthesis. Anchored PKA also regulates the activity of PP2A, whichpromotes PDE4D3 dephosphorylation, and RyR2, which enhances Ca²⁺mobilization from intracellular stores. This is proposed to induce theactivation of CaNAβ, which, in turn, dephosphorylates and activatesNFATc3 to promote hypertrophic gene transcription. Very highconcentrations of cAMP (in μM) also stimulate Epac1. This in turnactivates the GTPase Ras-related protein 1 (Rap1), which exerts aninhibitory effect on the MEK5-ERK5 pathway. In the absence of very highlocal cAMP, Epac1 is inactivated and the hypertrophic ERK5 pathwayde-repressed. Stimulation of endothelin-1 receptors (ET1Rs) activatesmAKAPβ-bound PLCε, which, in turn, promotes cardiomyocyte hypertrophyvia a signaling pathway that remains to be elucidated. B: activatedα₁-ARs and ET₁Rs stimulate the Rho-guanine nucleotide exchange factor(GEF) activity of AKAP-lymphoid blast crisis (AKAP-Lbc) through Gα₁₂.GTP-bound RhoA is released from the AKAP-Lbc complex and promotescardiomyocyte hypertrophy via a signaling pathway that remains to beelucidated. Activation of AKAP-Lbc-anchored PKA promotes thephosphorylation of the anchoring protein on serine-1565. This inducesthe recruitment of 14-3-3, which inhibits the Rho-GEF activity ofAKAP-Lbc. AKAP-Lbc also recruits PKCη and PKD. Upon stimulation by theGα_(q) phospholipase C pathway by α₁-ARs and ET₁Rs, PKCη becomesactivated and phosphorylates PKD. Active PKD phosphorylates histonedeacetylase 5 (HDAC5), causing its export from the nucleus. This favorsmyocyte-specific enhancer-binding factor 2 (MEF2)-dependent hypertrophicgene transcription. LIF-R, leukemia inhibitor factor receptor; IP3,inositol trisphosphate 1,4,5-trisphosphate.

DETAILED DESCRIPTION OF THE INVENTION

As discussed above, AKAP-based signaling complexes play a central rolein regulating physiological and pathological cardiac events. As such,the present inventors have examined inhibiting the signaling propertiesof individual AKAP signaling complexes using drugs that target uniqueprotein-protein interactions as an approach for limiting cardiacpathological processes. Such a therapeutic strategy offers an advantageover classical therapeutic approaches since it allows the selectiveinhibition of defined cellular responses.

Anchoring proteins including mAKAP are therapeutic targets for thetreatment of cardiac hypertrophy and heart failure. In particular, thepresent inventors have found that disrupting AKAP-mediatedprotein-protein interactions can be used to inhibit the ability of mAKAPto coordinate the activation of enzymes that play a central role inactivating key transcription factors that initiate the remodelingprocess leading to cardiac hypertrophy.

In particular, the inventors have found that type 3 ribosomal S6 kinase(RSK3) binds mAKAPβ directly via the unique N-terminal domain of RSK3,defining a novel enzyme-scaffold interaction. The inventors have foundthat anchored RSK3 regulates concentric cardiac myocyte growth,revealing an isoform-specific target for therapeutic intervention inpathologic cardiac hypertrophy. Delivery of such peptides that mightinhibit RSK3-mAKAPβ interaction can be enhanced by the use ofcell-penetrating sequences such as the transactivator of transcriptionpeptide and polyarginine tails, or conjugation with lipid-derived groupssuch as stearate. Stability may also be enhanced by the use ofpeptidomimetics [i.e., peptides with structural modifications in theoriginal sequence giving protection against exo- and endoproteaseswithout affecting the structural and functional properties of thepeptide. Alternatively, as shown in FIG. 41, peptides can be deliveredby intracellular expression via viral-based gene therapy vectors.

The inventors have also found that small molecule disruptors can be usedto target specific interaction within AKAP-based complexes. Smallmolecule disruptors can be identified by combining rational design andscreening approaches. Such compounds can be designed to target-specificbinding surfaces on AKAPs, to disrupt the interaction between AKAPs andPKA in cardiomyocytes and to enhance the contractility of intact heartsfor the treatment of chronic heart failure.

The present invention relates to methods of treating any cardiaccondition, which is initiated through the interaction of RSK3 andmAKAPβ. Such cardiac dysfunction can result in signs and symptoms suchas shortness of breath and fatigue, and can have various causes,including, but not limited to hypertension, coronary artery disease,myocardial infarction, valvular disease, primary cardiomyopathy,congenital heart disease, arrhythmia, pulmonary disease, diabetes,anemia, hyperthyroidism and other systemic diseases.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, “Molecular Cloning:A Laboratory Manual” (4th Ed., 2012); “Current Protocols in MolecularBiology” Volumes I-III [Ausubel, R. M., ed. (1994)]; “Cell Biology: ALaboratory Handbook” Volumes I-III [J. E. Celis, 3rd ed. (2005))];“Current Protocols in Immunology” Volumes I-III [Coligan, J. E., ed.(2005)]; “Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “NucleicAcid Hybridization” [B. D. Hames & S. J. Higgins eds. (1985)];“Transcription And Translation” [B. D. Hames & S. J. Higgins, eds.(1984)]; “Animal Cell Culture” [R. I. Freshney, ed. (1986)];“Immobilized Cells And Enzymes” [IRL Press, (1986)]; B. Perbal, “APractical Guide To Molecular Cloning” (1984); C. Machida, “Viral Vectorsfor Gene Therapy: Methods and Protocols” (2010); J. Reidhaar-Olson andC. Rondinone, “Therapeutic Applications of RNAi: Methods and Protocols”(2009).

The following definitions and acronyms are used herein:

ANF atrial natriuretic factor

CTKD C-terminal kinase domain

ERK extracellular signal-regulated kinase

FBS fetal bovine serum

GFP green fluorescent protein

Iso isoproterenol

LIF leukemia inhibitory factor

mAKAP muscle A-kinase anchoring protein

NTKD N-terminal kinase domain

PDK1 3′phosphoinositide-dependent kinase 1

PE phenylephrine

RBD RSK binding domain

RSK p90 ribosomal S6 kinase

siRNA small interfering RNA oligonucleotide

TAC transverse aortic constriction

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described. Generally, nomenclatures utilized inconnection with, and techniques of, cell and molecular biology andchemistry are those well known and commonly used in the art. Certainexperimental techniques, not specifically defined, are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification. For purposes of theclarity, following terms are defined below.

The present invention recognizes that the interaction of RSK3 and mAKAPβmediates various intracellular signals and pathways which lead tocardiac myocyte hypertrophy and/or dysfunction. As such, the presentinventors have discovered various methods of inhibiting that interactionin order to prevent and/or treat cardiac myocyte hypertrophy and/ordysfunction.

Thus, the present invention includes a method for protecting the heartfrom damage, by administering to a patient at risk of such damage, apharmaceutically effective amount of a composition, which inhibits theinteraction of RSK3 and mAKAPβ. It should be appreciated that “apharmaceutically effective amount” can be empirically determined basedupon the method of delivery, and will vary according to the method ofdelivery.

The invention also relates to a method of treating heart disease, byadministering to a patient a pharmaceutically effective amount of acomposition, which inhibits the interaction of RSK3 and mAKAPβ.

The invention also relates to compositions which inhibit the interactionof RSK3 and mAKAPβ. In particular embodiments, these inhibitingcompositions or “inhibitors” include peptide inhibitors, which can beadministered by any known method, including by gene therapy delivery. Inother embodiments, the inhibitors can be small molecule inhibitors.

Specifically, the present invention is directed to methods andcompositions for treating or protecting the heart from damage, byadministering to a patient at risk of such damage, a pharmaceuticallyeffective amount of a composition which (1) inhibits the interaction ofRSK3 and mAKAPβ; (2) inhibits the activity of RSK3 and mAKAPβ; or (3)inhibits the expression of RSK3 and mAKAPβ.

The invention also relates to methods of treating or protecting theheart from damage, by administering to a patient at risk of such damage,a pharmaceutically effective amount of a composition which inhibits acellular process mediated by the anchoring of RSK3 through itsN-terminal domain.

In one embodiment, the composition includes an RSK3 peptide. In apreferred embodiment, the RSK3 peptide is obtained from the aminoterminus of the RSK3 amino acid sequence. In a particularly preferredembodiment, the RSK3 peptide is amino acids 1-42 of the RSK3 amino acidsequence.

In another embodiment, the composition includes a small interfering RNAsiRNA that inhibits the expression of either or both of RSK3 and mAKAPβ.

The composition of the invention can be administered directly or can beadministered using a viral vector. In a preferred embodiment, the vectoris adeno-associated virus (AAV).

In another embodiment, the composition includes a small moleculeinhibitor. In preferred embodiments, the small molecule is SL0101 orBI-D1870.

In another embodiment, the composition includes a molecule that inhibitsthe binding, expression or activity of mAKAPβ. In a preferredembodiment, the molecule is a mAKAPβ peptide. The molecule may beexpressed using a viral vector, including adeno-associated virus (AAV).

In yet another embodiment, the composition includes a molecule thatinterferes with RSK3-mediated cellular processes. In preferredembodiments, the molecule interferes with the anchoring of RSK3 throughits N-terminal domain.

The invention also relates to diagnostic assays for determining apropensity for heart disease, wherein the binding interaction of RSK3and mAKAPβ is measured, either directly, or by measuring a downstreameffect of the binding of RSK3 and mAKAPβ. The invention also provides atest kit for such an assay.

In still other embodiments, the inhibitors include any molecule thatinhibits the expression of RSK3 and mAKAPβ, including antisense RNA,ribozymes and small interfering RNA (siRNA).

The invention also includes an assay system for screening of potentialdrugs effective to inhibit the expression and/or binding of RSK3 andmAKAPβ. In one instance, the test drug could be administered to acellular sample with the RSK3 and mAKAPβ, or an extract containing theRSK3 and mAKAPβ, to determine its effect upon the binding activity ofthe RSK3 and mAKAPβ, by comparison with a control. The invention alsoprovides a test kit for such an assay.

In preparing the peptide compositions of the invention, all or part ofthe RSK3 (FIG. 2) or mAKAPβ (FIG. 3) amino acid sequence may be used. Inone embodiment, the amino-terminal region of the RSK3 protein is used asan inhibitor. Preferably, at least 10 amino acids of the RSK3 aminoterminus are used. More preferably, about 18 amino acids of the RSK3amino terminus are used. Most preferably, amino acids from about 1-42 ofthe RSK3 amino terminus are used.

In other embodiments, at least 10 amino acids of the mAKAPβ sequence areused. More preferably, at least 25 amino acids of the mAKAPβ sequenceare used. Most preferably, peptide segments from amino acids 1694-1833of the RSK3 amino terminus are used.

It should be appreciated that various amino acid substitutions,deletions or insertions may also enhance the ability of the inhibitingpeptide to inhibit the interaction of RSK3 and mAKAPβ. A substitutionmutation of this sort can be made to change an amino acid in theresulting protein in a non-conservative manner (i.e., by changing thecodon from an amino acid belonging to a grouping of amino acids having aparticular size or characteristic to an amino acid belonging to anothergrouping) or in a conservative manner (i.e., by changing the codon froman amino acid belonging to a grouping of amino acids having a particularsize or characteristic to an amino acid belonging to the same grouping).Such a conservative change generally leads to less change in thestructure and function of the resulting protein. A non-conservativechange is more likely to alter the structure, activity or function ofthe resulting protein. The present invention should be considered toinclude sequences containing conservative changes, which do notsignificantly alter the activity, or binding characteristics of theresulting protein.

The following is one example of various groupings of amino acids:

Amino acids with nonpolar R groups: Alanine, Valine, Leucine,Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine.

Amino acids with uncharged polar R groups: Glycine, Serine, Threonine,Cysteine, Tyrosine, Asparagine, Glutamine.

Amino acids with charged polar R groups (negatively charged at pH 6.0):Aspartic acid, Glutamic acid.

Basic amino acids (positively charged at pH 6.0): Lysine, Arginine,Histidine (at pH 6.0).

Another grouping may be those amino acids with phenyl groups:Phenylalanine, Tryptophan, Tyrosine.

Another grouping may be according to molecular weight (i.e., size of Rgroups): Glycine (75), Alanine (89), Serine (105), Proline (115), Valine(117), Threonine (119), Cysteine (121), Leucine (131), Isoleucine (131),Asparagine (132), Aspartic acid (133), Glutamine (146), Lysine (146),Glutamic acid (147), Methionine (149), Histidine (at pH 6.0) (155),Phenylalanine (165), Arginine (174), Tyrosine (181), Tryptophan (204).

Particularly preferred substitutions are:

-   -   Lys for Arg and vice versa such that a positive charge may be        maintained;    -   Glu for Asp and vice versa such that a negative charge may be        maintained;    -   Ser for Thr such that a free —OH can be maintained; and    -   Gln for Asn such that a free NH₂ can be maintained.

Amino acid substitutions may also be introduced to substitute an aminoacid with a particularly preferable property. For example, a Cys may beintroduced a potential site for disulfide bridges with another Cys. AHis may be introduced as a particularly “catalytic” site (i.e., His canact as an acid or base and is the most common amino acid in biochemicalcatalysis). Pro may be introduced because of its particularly planarstructure, which induces-turns in the protein's structure. Two aminoacid sequences are “substantially homologous” when at least about 70% ofthe amino acid residues (preferably at least about 80%, and mostpreferably at least about 90 or 95%) are identical, or representconservative substitutions.

Likewise, nucleotide sequences utilized in accordance with the inventioncan also be subjected to substitution, deletion or insertion. Wherecodons encoding a particular amino acid are degenerate, any codon whichcodes for a particular amino acid may be used. In addition, where it isdesired to substitute one amino acid for another, one can modify thenucleotide sequence according to the known genetic code.

Two nucleotide sequences are “substantially homologous” when at leastabout 70% of the nucleotides (preferably at least about 80%, and mostpreferably at least about 90 or 95%) are identical.

The term “standard hybridization conditions” refers to salt andtemperature conditions substantially equivalent to 5×SSC and 65 C forboth hybridization and wash. However, one skilled in the art willappreciate that such “standard hybridization conditions” are dependenton particular conditions including the concentration of sodium andmagnesium in the buffer, nucleotide sequence length and concentration,percent mismatch, percent formamide, and the like. Also important in thedetermination of “standard hybridization conditions” is whether the twosequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standardhybridization conditions are easily determined by one skilled in the artaccording to well known formulae, wherein hybridization is typically10-20 C below the predicted or determined T_(m) with washes of higherstringency, if desired.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human.

The phrase “therapeutically effective amount” is used herein to mean anamount sufficient to prevent, and preferably reduce by at least about 30percent, more preferably by at least 50 percent, most preferably by atleast 90 percent, a clinically significant change in a cardiac myocytefeature.

The preparation of therapeutic compositions which contain polypeptides,analogs or active fragments as active ingredients is well understood inthe art. Typically, such compositions are prepared as injectables,either as liquid solutions or suspensions, however, solid forms suitablefor solution in, or suspension in, liquid prior to injection can also beprepared. The preparation can also be emulsified. The active therapeuticingredient is often mixed with excipients which are pharmaceuticallyacceptable and compatible with the active ingredient. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanol,or the like and combinations thereof. In addition, if desired, thecomposition can contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents which enhance theeffectiveness of the active ingredient.

A polypeptide, analog or active fragment, as well as a small moleculeinhibitor, can be formulated into the therapeutic composition asneutralized pharmaceutically acceptable salt forms. Pharmaceuticallyacceptable salts include the acid addition salts (formed with the freeamino groups of the polypeptide or antibody molecule) and which areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed from the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine, and the like.

The therapeutic compositions of the invention are conventionallyadministered intravenously, as by injection of a unit dose, for example.The term “unit dose” when used in reference to a therapeutic compositionof the present invention refers to physically discrete units suitable asunitary dosage for humans, each unit containing a predetermined quantityof active material calculated to produce the desired therapeutic effectin association with the required diluent; i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's immune system to utilize the active ingredient, and degree ofinhibition of RSK3-mAKAPβ binding desired. Precise amounts of activeingredient required to be administered depend on the judgment of thepractitioner and are peculiar to each individual. However, suitabledosages may range from about 0.1 to 20, preferably about 0.5 to about10, and more preferably one to several, milligrams of active ingredientper kilogram body weight of individual per day and depend on the routeof administration. Suitable regimes for initial administration andbooster shots are also variable, but are typified by an initialadministration followed by repeated doses at one or more hour intervalsby a subsequent injection or other administration. Alternatively,continuous intravenous infusion sufficient to maintain concentrations often nanomolar to ten micromolar in the blood are contemplated.

Because of the necessity for the inhibitor to reach the cytosol, apeptide in accordance with the invention may need to be modified inorder to allow its transfer across cell membranes, or may need to beexpressed by a vector which encodes the peptide inhibitor. Likewise, anucleic acid inhibitor (including siRNAs and antisense RNAs) can beexpressed by a vector. Any vector capable of entering the cells to betargeted may be used in accordance with the invention. In particular,viral vectors are able to “infect” the cell and express the desired RNAor peptide. Any viral vector capable of “infecting” the cell may beused. A particularly preferred viral vector is adeno-associated virus(AAV).

With respect to small molecule inhibitors, any small molecule thatinhibits the interaction of RSK3 and mAKAPβ may be used. In addition,any small molecules that inhibit the activity of RSK3 and/or mAKAPβ maybe used. Particularly preferred small molecules include BI-D1870,available from Enzo Life Sciences

and SL0101, available from Millipore:

Small molecules with similar structures and functionalities can likewisebe determined by rational and screening approaches.

Likewise, any small molecules that inhibit the expression of RSK3 and/ormAKAPβ may be used.

In yet more detail, the present invention is described by the followingitems which represent preferred embodiments thereof:

-   -   1. A method of protecting the heart from damage, by        administering to a patient at risk of such damage, a        pharmaceutically effective amount of a composition which        inhibits the interaction of RSK3 and mAKAPβ.    -   2. The method of item 1, wherein the composition is a RSK3        peptide.    -   3. The method of item 2, wherein the RSK3 peptide is obtained        from the amino terminus of the RSK3 amino acid sequence.    -   4. The method of item 3, wherein the RSK3 peptide is amino acids        1-42 of the RSK3 amino acid sequence.    -   5. The method of item 2, wherein the polypeptide is administered        directly.    -   6. The method of item 2, wherein the polypeptide is administered        using a viral vector.    -   7. The method of item 6, wherein the vector is adeno-associated        virus (AAV).    -   8. The method of item 1, wherein the composition is a small        interfering RNA (siRNA) that inhibits the expression of RSK3.    -   9. A method of treating heart disease, by administering to a        patient a pharmaceutically effective amount of a composition        which inhibits the interaction of RSK3 and mAKAPβ.    -   10. The method of item 9, wherein the composition is a RSK3        peptide.    -   11. The method of item 10, wherein the RSK3 peptide is obtained        from the amino terminus of the RSK3 amino acid sequence.    -   12. The method of item 11, wherein the RSK3 peptide is amino        acids 1-42 of the RSK3 amino acid sequence.    -   13. The method of item 9, wherein the polypeptide is        administered directly.    -   14. The method of item 9, wherein the polypeptide is        administered using a viral vector.    -   15. The method of item 14, wherein the vector is        adeno-associated virus (AAV).    -   16. The method of item 19, wherein the composition is a small        interfering RNA (siRNA) that inhibits the expression of RSK3.    -   17. A composition comprising a molecule which inhibits the        interaction of RSK3 and mAKAPβ.    -   18. The composition of item 17, wherein the molecule is a        peptide.    -   19. The composition of item 18, wherein the molecule is a RSK3        peptide.    -   20. The composition of item 19, wherein the RSK3 peptide is        obtained from the amino terminus of the RSK3 amino acid        sequence.    -   21. The composition of item 20, wherein the RSK3 peptide is        amino acids 1-42 of the RSK3 amino acid sequence.    -   22. The composition of item 17, wherein the molecule is        expressed using a viral vector.    -   23. The composition of item 22, wherein the vector is        adeno-associated virus (AAV).    -   24. The composition of Item 17, wherein the molecule is a small        molecule.    -   25. A method of treating or preventing heart disease, by        administering to a patient a pharmaceutically effective amount        of a composition which inhibits the activity of RSK3.    -   26. The composition of item 25, wherein the small molecule is        SL0101 or BI-D1870.    -   27. A method of treating or preventing heart disease, by        administering to a patient a pharmaceutically effective amount        of a composition which inhibits the activity of mAKAPβ.    -   28. The method of item 27, wherein the composition is a mAKAPβ        peptide.    -   29. The composition of item 28, wherein the mAKAPβ peptide is        amino acids 1694-1833 of the mAKAPβ amino acid sequence.    -   30. The composition of item 28, wherein the mAKAPβ peptide is        amino acids 1735-1833 of the mAKAPβ amino acid sequence.    -   31. The composition of item 27, wherein the molecule is        expressed using a viral vector.    -   32. The composition of item 31, wherein the vector is        adeno-associated virus (AAV).

The following examples are provided to aid the understanding of thepresent invention, the true scope of which is set forth in the appendedclaims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

EXAMPLES

The compositions and processes of the present invention will be betterunderstood in connection with the following examples, which are intendedas an illustration only and not limiting of the scope of the invention.Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art and such changes and modificationsincluding, without limitation, those relating to the processes,formulations and/or methods of the invention may be made withoutdeparting from the spirit of the invention and the scope of the appendedclaims.

Example 1 Methods

Reagents

Commercial antibodies and oligonucleotides are listed in FIG. 19 andFIG. 20. The commercially available RSK3 antibodies were of varyingspecificity (FIG. 21). Additional reagents and detailed methods areprovided.

RSK3^(−/−) Mouse

All experiments involving animals were approved by the InstitutionalAnimal Care and Use Committee at the University of Miami. ConstitutiveRSK3 knockout mice were backcrossed to C57BL/6 mice over 10 generations.All experiments were performed with littermate controls and mice thatwere 8 to 10 weeks of age at the beginning of the study. Transverseaortic constriction was performed as previously described (Rockman1991), and isoproterenol infusion was via Alzet 2002 osmotic pumps(Durect). Echocardiography was performed under isoflurane anesthesia ona Vevo 770 High-Resolution Imaging System (VisualSonics).

RNA Assays

mRNA species were assayed using NanoString technology, a direct andmultiplexed measurement of gene expression without amplification, usingfluorescent molecular bar codes and single-molecule imaging to identifyand count multiple transcripts in a single reaction; 100 ng of RNA washybridized in solution to a target-specific code set overnight at 65°C., and individual mRNAs were counted using a NanoString DigitalAnalyzer.

Statistics

For all experiments, n refers to the number of individual mice orindividual myocyte preparations. All data are expressed as mean±SEM. Pvalues were calculated using Student t tests and are not corrected formultiple comparisons. Repeated symbols represent P values of differentorders of magnitude (i.e., P<0.05, P<0.005, and others). All datasetsinvolving multiple comparisons for which P values are provided also weresignificant by ANOVA (α=0.05).

Results

mAKAPβ: A Scaffold for RSK

The inventors have previously published that RSK proteins and activityare associated with mAKAPa complexes in the brain (Michel 2005). Theinventors now show that RSK also is associated with mAKAPβ in cardiacmyocytes (FIG. 4B). To determine whether mAKAP preferentially binds aspecific RSK isoform, hemagglutinin (HA)-tagged RSK family members wereco-expressed with mAKAP in HEK293 cells. In contrast to RSK1 and RSK2,RSK3 robustly mediated the coimmunoprecipitation of both mAKAPa andmAKAPβ (FIG. 4C). RSK family members are similar in primary sequencewith the exception of the extreme N-terminal and C-terminal domains anda small region after the hydrophobic motif (FIG. 4A). Consistent withthe selective binding of RSK3 to the scaffold, the N-terminal domain ofRSK3 bound mAKAPβ (FIG. 4D).

RSK3 Function in Neonatal Cardiac Myocytes

RSK family members can be activated in most cell types by ERK, but notc-Jun N-terminal kinases or p38 (Anjum 2008). ERK phosphorylation ispermissive for PDK1 phosphorylation of the RSK N-terminal kinase domain,such that PDK1 phosphorylation of RSK S²¹⁸ is indicative of fullactivation of the enzyme (FIG. 4A). To show that ERK activates RSK incardiac myocytes, the inventors treated neonatal rat ventricularmyocytes with different hypertrophic agonists and mitogen-activatedprotein kinase pathway inhibitors and detected RSK activation using apan-RSK S²¹⁸ phosphor-specific antibody (FIGS. 21 and 22). Theα-adrenergic stimulation with phenylephrine (PE) induced RSKphosphorylation 3-fold by both MEK1/2-dependent (that activates ERK1/2)and MEK5-dependent (that activates ERK5) mechanisms (FIG. 22). Moreover,MEK1/2 inhibition reduced RSK baseline phosphorylation. c-Jun N-terminalkinase and p38 inhibition did not affect PE activation and, in fact,variably increased baseline RSK phosphorylation. Fetal bovine serum andleukemia inhibitory factor also increased the level of activated RSK,but that occurred more so because of an increase in total RSK proteinexpression than because of ERK phosphorylation.

Similar results were found for HA-tagged RSK3 (FIG. 5A). Acute PEtreatment induced the phosphorylation of HA-RSK3 ERK (S³⁶⁰) and PDK1(S²¹⁸) sites through both MEK1/2-dependent and MEK5-dependent signaling.Together, these results confirmed that in cardiac myocytes ERK isresponsible for RSK activation.

The inventors have previously demonstrated that mAKAPβ complexes arerequired for the hypertrophy of cultured myocytes (Li 2010, Pare 2005,Dodge-Kafka 2005). Therefore, the inventors proposed the hypothesis thatRSK3 signaling is a major determinant of cardiac myocyte growth.Neonatal myocytes were transfected with small interfering RNAoligonucleotides (siRNA) that diminished RSK3 mRNA and protein levelsby >75% (FIG. 5B). RSK3 siRNA did not induce the apoptosis of myocytescultured either in the absence or in the presence of serum (FIG. 5C).Importantly, in the presence of α-adrenergic stimulation, RSK3 siRNAinhibited morphologic hypertrophy by 34% and atrial natriuretic factorexpression completely (FIGS. 5D-5F and FIG. 23). In addition, RSK3 siRNAhad smaller, but detectable, effects on leukemia inhibitory factor andfetal bovine serum-stimulated hypertrophy. The results obtained by RSK3RNA interference were confirmed with a second distinct RSK3 siRNA.

Endogenous RSK3 proteins are expressed at a relatively low level incardiac myocytes compared with the other RSK family members and areinduced in expression by long-term PE treatment (FIG. 24C). As a result,RSK3 RNA interference did not affect the level of total RSK in themyocyte, only diminishing the RSK3 detected after immunoprecipitationwith a specific RSK3 antibody (FIG. 5B and FIG. 24C). In controlexperiments, the inhibition of PE-induced hypertrophy by the RSK3 siRNAwas rescued by the expression of recombinant HA-tagged human RSK3, butnot by an inactive HA-RSK3 S²¹⁸A mutant (FIGS. 24A and 24B). Remarkably,in these experiments, the cross-section area of unstimulated myocyteswas increased by adenoviral-based expression of wild-type HA-RSK3 enzymeat a level comparable with that of endogenous RSK3 in PE-treated cellswithout affecting total RSK levels. Finally, to confirm that RSKactivity was important for neonatal myocyte hypertrophy, the inventorsused the pan-RSK inhibitors BI-D1870 (FIG. 6 and FIG. 25) and SL0101(FIG. 26) (Smith 2005, Sapkota 2007, Malone 2005), finding that, likeRSK3 siRNA, these compounds inhibited agonist-induced myocytehypertrophy.

High-Affinity RSK3 Binding Domain in mAKAP

The inventors considered that the requirement for RSK3 in myocytehypertrophy was attributable to the association of RSK3 with specificsignaling complexes. To address this hypothesis, the inventors definedthe mAKAP domains responsible for RSK3 binding. HA-tagged RSK3 wasco-expressed in heterologous cells with myc-tagged mAKAPβ fragments andcoimmunoprecipitated using a myc-tag antibody (FIGS. 7A and 7B). RSK3preferentially associated with mAKAP amino acid residues 1286 to 1833,although it also weakly associated with mAKAP 245 to 587 and 525 to1286. Consistent with this result, RSK3 binding to a full-length mAKAPβprotein with an internal deletion of residues 1257 to 1886 was reducedby >85%. Further mapping showed that the main RSK3 binding domain (RBD)of mAKAP mapped to a fragment encompassing residues1694 to 1833 (FIG.7C). Accordingly, RSK3 bound poorly to a full-length mAKAPβ protein withan internal deletion of residues 1701 to 1800 (FIG. 7D). As shown, theunique N-terminal domain of RSK3 bound full-length mAKAPβ (FIG. 4D). ThemAKAP RBD also bound HA-RSK3 1 to 42 (FIG. 7E), but not to theN-terminally truncated RSK3 mutant (HA-RSK3 DN30) or the HA-taggedfull-length RSK2 (FIG. 7F). These results imply that the mAKAP RBD isresponsible for the selective binding of RSK3 to mAKAP.

The inventors next tested whether mAKAP-RSK3 binding is direct (FIG.7G). The binding of bacterially expressed His-tagged mAKAP 1286 to 1833and full-length RSK3 was analyzed by surface plasmon resonance. Thebinding was direct and of high affinity (nanomolar K_(D)). The inventorspreviously reported that once activated, RSK3 binds mAKAPa less well incells (Michele 2005). Interestingly, previous RSK3 phosphorylation byeither ERK or both ERK and PDK1 decreased the RSK3 binding affinity formAKAP 5-fold and 8-fold, respectively, through a decrease in theassociation rate constant.

Disruption of RSK3 Anchoring Inhibits Neonatal Myocyte Hypertrophy

The identification of the high-affinity mAKAP RBD provided theopportunity to test whether anchoring of RSK3 is important for itsfunction. When expressed in neonatal myocytes, a green fluorescentprotein-mAKAP RBD fusion protein competed the association of endogenousRSK3 and mAKAPβ (FIG. 8A). Expression of green fluorescent protein-mAKAPRBD fusion protein markedly inhibited PE-induced hypertrophy (FIG.8B-5D), similar to RSK3 siRNA (FIG. 5). Together, these results implythat RSK3 anchored to scaffolds through its unique N-terminal domain isrequired for the hypertrophy of cultured myocytes.

Role of RSK3 in Cardiac Hypertrophy In Vivo

The results obtained in vitro suggested that active RSK3 contributes tothe development of pathologic myocyte hypertrophy. Further evidencesupporting this hypothesis was obtained using a new RSK3 knockout mouse.By homologous recombination, stop codons were inserted into the secondexon of the RSK3 (Rps6ka2) gene encoding the ATP-binding motif of theN-terminal kinase domain (Hanks 1988), resulting in the constitutiveabsence of RSK3 protein in homozygous null mice (FIG. 27). In general,RSK3^(−/−) mice appeared normal in morphology, were bred according toMendelian genetics (FIG. 12), and exhibited no excess mortality up to 6months of age. Before any stress, the RSK3^(−/−) mice had generallynormal cardiac function, with the only measureable difference fromwild-type littermates being a slight increase in left ventricularinternal dimensions detected by echocardiography (FIGS. 13 and 28).

The inventors tested whether RSK3 is required for compensated cardiachypertrophy by subjecting the RSK3^(−/−) mice to pressure overload for 2weeks (FIG. 9A). By echocardiography, transverse aortic constriction(TAC) induced a 36% increase in posterior wall thickness in wild-typemice, but only a 16% increase in RSK3^(−/−) mice (FIGS. 10 and 28). Thedecreased hypertrophy was not accompanied by a change in contractility(fractional shortening). Postmortem gravimetric analysis showed that thecorresponding increase in biventricular weight after TAC was similarlydiminished in the knockout mice (48% for RSK3^(+/+) vs. 26% forRSK3^(−/−) mice; FIG. 14). TAC primarily induces concentric growth ofcardiac myocytes.

Inspection of wheat germ agglutinin-stained heart sections revealed thatconsistent with these results, RSK3 knockout attenuated the TAC-inducedincrease in myocyte transverse cross-section area by ˜46% (FIGS. 9B and9C). Proportional results were obtained by morphometric analysis ofadult cardiac myocytes isolated from the TAC mice (FIG. 9D-9G).

To characterize the RSK3^(−/−) cardiac phenotype at a molecular level,the inventors surveyed for differences in the cardiac expression of 30genes encoding proteins involved either in cardiac remodeling or inhypertrophic signaling (FIG. 11). Approximately two-thirds of the genesin our panel were significantly increased or decreased in expression byTAC. In general, the changes in expression were attenuated by RSK3knockout. For example, TAC-induced atrial natriuretic factor expressionwas dramatically inhibited in RSK3^(−/−) mice, consistent with theresults obtained for PE-treated neonatal myocytes. Although after 2weeks of pressure overload the small increases in cellular apoptosis andinterstitial fibrosis detectable by histology for wild-type mice did notreach significance when compared with sham-operated controls, thesesigns of remodeling tended to be less in the knockout mice (8.2±2.0 vs.4.2±1.0×10⁻⁴ TUNEL-positive nuclei and 0.49%±0.18% vs. 0.29%±0.11%collagen staining for wild-type and RSK3^(−/−) TAC hearts,respectively). Interestingly, 2 genetic markers of fibrosis that weresignificantly induced in TAC wild-type mice, transforming growth factorβ2 and collagen VI α1 (Yang 2012), were attenuated in expression by RSK3knockout (FIG. 11).

To further explore the role of RSK3 in cardiac hypertrophy, theinventors used a second in vivo pathological stressor, chronicisoproterenol (Iso) infusion via subcutaneous osmotic pumps, and aphysiological stressor, chronic exercise via swimming. Although Isoinfusion resulted in a minor increase in ventricular wall thickness byechocardiography (FIG. 15), at the cellular level Iso significantlyinduced myocyte growth in width in a RSK3-dependent manner as measuredby histology and after myocyte isolation (FIG. 29). Unlike TAC, Isoinfusion also induced eccentric growth, as evidenced by increasedmyocyte length and ventricular dilation by echocardiography (FIGS. 15and 16). This eccentric growth was not inhibited by RSK3 knockout.Together with the TAC data, these results demonstrate that RSK3contributes to the induction of concentric myocyte hypertrophy inpathologic conditions.

Finally, RSK3^(−/−) mice were exercised by swimming. As expected(Perrino 2006), after swimming, wild-type mice exhibited a decreasedresting heart rate (consistent with improved physical conditioning) andincreased left ventricular internal dimensions (FIGS. 17 and 28). Afterexercise, there were no significant differences between RSK3 knockoutand wild-type mice detectable by echocardiography, and the cohortsexhibited a similar increase in biventricular weight indexed by bodyweight (6% and 7%, respectively; FIG. 18).

Detailed Methods

Reagents:

Commercial antibodies are listed in FIG. 19. Secondary antibodiesincluded horseradish peroxidase (HRP)-conjugated donkey secondaryantibodies (Jackson ImmunoResearch) and Alexa dye-conjugated donkeysecondary antibodies (Invitrogen). Monoclonal 211, polyclonal VO54, VO56and OR010 mAKAP antibodies were as previously described and areavailable through Covance Research Products (Kehat 2010). OR42 and OR43rabbit anti-RSK3 antisera were generated using bacterially-expressedHis-tagged RSK3 (full-length) and affinity purified usingantigen-coupled Affigel resin (Biorad). FL099 and FL100 rabbitanti-mAKAP antisera were generated using bacterially-expressedGST-tagged mAKAP 245-340. Oligonucleotides are listed in FIG. 20. Otherreagents included: BIX02189—Boehringer Ingelheim Pharmaceuticals;PD0325901, SB103580, SL0101, and SP600125—EMD Chemicals Inc.

All adenovirus were constructed using the pTRE shuttle vector and theAdeno-X Tet-off System (Clontech) and purified after amplification usingVivapure AdenoPACK kits (Sartorius Stedim). These adenovirusesconditionally express recombinant protein when co-infected withtetracycline transactivator-expressing virus (adeno-tTA for “tet-off” orreverse tTA for “tet-on”). HA-tagged RSK and MSK2 expression plasmidsacquired from Dario Alessi and John Blenis (McKinsey 2007, Anjum 2008,Sadoshima 1995) and myc-tagged mAKAP mammalian expression vectors(pCDNA3.1 (−) myc-his) and adenovirus were as previously described(Kodama 2000, Takeishi 2002). GFP-RBD was expressed using a pEGFP-basedplasmid. Bacterial expression vectors for mAKAP and RSK3 wereconstructed using pET30 and pGEX-4T parent vectors, and proteins werepurified using His-bind (Novagen) and Glutathione Uniflow Resins(Clontech).

Neonatal Rat Myocytes Isolation and Culture:

1-3 day old Sprague-Dawley rats were decapitated and the excised heartsplaced in 1×ADS Buffer (116 mmol/L NaCl, 20 mmol/L HEPES, 1 mmol/LNaH₂PO₄, 5.5 mmol/L glucose, 5.4 mmol/L KCl, 0.8 mmol/L MgSO₄, pH 7.35).The atria were carefully removed and the blood washed away. Theventricles were minced and incubated with 15 mL 1×ADS Buffer containing3.3 mg type II collagenase (Worthington, 230 U/mg) and 9 mg Pancreatin(Sigma) at 37° C. while shaking at 80 RPM. After 15 minutes, thedissociated cardiac myocytes were separated by centrifugation at 50×gfor 1 minute, resuspended in 4 mL horse serum and incubated 37° C. withoccasional agitation. The steps for enzymatic digestion and isolation ofmyocytes were repeated 10-12 times to maximize yield. The myocytes werepooled and spun down again at 50×g for 2 minutes and resuspended inMaintenance Medium (DMEM:M199, 4:1) supplemented with 10% horse serumand 5% fetal bovine serum. To remove any contaminating fibroblasts, thecells were pre-plated for 1 hour before plating on gelatin-coated tissueculture plasticware. This procedure yields >90% pure cardiac myocytes.After 1 day in culture, the media was changed to maintenance mediumcontaining 0.1 mmol/L bromodeoxyuridine to suppress fibroblast growth.

Experiments were initiated 1 day after myocyte isolation. Adenoviralinfection was performed by addition of adenovirus (multiplicity ofinfection=5-50) to the media. Plasmids and siRNA oligonucleotides weretransfected using Transfast (Promega) and Dharmafect (Thermofisher),respectively, as recommended by the manufacturers using cells culturedin maintenance medium supplemented with 4% horse serum. Starting the dayafter gene transduction, the cells were treated for as long as 2 days,as indicated for each experiment.

Immunoprecipitations:

HEK293 and COS-7 cells were transfected with Lipofectamine 2000(Invitrogen) or Polyethylenimine “Max” (Polysciences). Cells (includingmyocytes) were lysed in buffer (20 mmol/L HEPES, pH 7.4, 150 mmol/LNaCl, 5 mmol/L EDTA, 0.5% Triton, 50 mmol/L NaF, 1 mmol/L sodiumorthovanadate, 1 mmol/L DTT, and protease inhibitors). Aftercentrifugation at 10,000×g for 10 minutes at 4° C., the clarifiedextracts were used for immunoprecipitation using appropriate antibodies(10 μg purified antibody or 1-5 μL whole serum) and 20 μL protein Gsepharose (Millipore, Fastflow) for 3 hours to overnight at 4° C. Thebeads were washed 3-5 times with lysis buffer, and theimmunoprecipitated proteins were eluted with 1× Laemmli buffer forwestern blotting. Western blots were developed using horseradishperoxidase-conjugated donkey secondary antibodies, Supersignal WestChemiluminescent Substrates (Thermo Scientific) and X-ray film or aFujifilm LAS-3000 imaging system.

Immunocytochemistry:

Cultured neonatal cardiomyocytes on plastic coverslips were fixed in3.7% formaldehyde in PBS, permeabilized with 0.3% Triton X-100 in PBS,and blocked with PBS containing 0.2% BSA and 1% horse serum for 1 hour.The slides were then sequentially incubated for 1 hour with primary andAlexa fluorescent dye-conjugated specific-secondary antibodies(Invitrogen, 1:1000) diluted in blocking buffer. The slips were washedthree times with blocking buffer. 1 μg/mL Hoechst 33258 was included inthe last wash stop to label nuclei. Slides were sealed in SlowFade Goldantifade buffer (Invitrogen) for fluorescent microscopy. Wide-fieldimages were acquired using a Leica DMI 6000 Microscope.

Surface Plasmon Resonance:

SPR analysis was performed using a BIAcore T100. 200 resonance unitsHis-tagged mAKAP 1286-1833 were covalently immobilized using NHS(N-hydroxysuccinamide) and EDC[1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide] (Biacore aminecoupling kit) to the surface of a sensor chip (BIAcore type CM5).His-RSK3 analytes (6.25-200 nmol/L) in HBS buffer (10 mmol/L Hepes, pH7.4, 150 mmol/L NaCl, and 0.005% Surfactant P20) were injected at a flowrate of 30 μL/min for 5 minutes, followed by buffer alone for another 5minutes. Sensorgrams were processed by BIAcore T100 evaluation software.

For phosphorylated His-RSK3, 20 μg His-RSK3 was phosphorylated with 2 μgERK2 (Millipore, 14-550) and/or PDK1 (Sigma, P7498) for 5 hours inkinase buffer (20 mmol/L MOPS, pH 7.2, 25 mmol/L β-glycerol phosphate, 5mmol/L EGTA, 1 mmol/L sodium orthovanadate, 1 mmol/L DTT) with 0.5mmol/L MgATP. Phospho-His-RSK3 was purified using His-binding beads andconcentrated before use.

Generation of RSK3^(−/−) Mouse:

All experiments involving animals were approved by the InstitutionalAnimal Care and Use Committee at the University of Miami. Constitutiveknock-out mice were generated using a targeting vector that insertedinto Exon 2 a neomycin resistance gene (PGKneo) flanked by loxp sites(FIG. 27). Targeted 129SvJ ES cells were injected into C57BL/6Jblastocysts. PGKneo was removed by crossing mutant mice withB6.C-Tg(CMV-cre)1Cgn/J (The Jackson Laboratory). RSK^(+/−) Mice wereselected for loss of the cre transgene and backcrossed to C57BL/6 miceover 10 generations. All experiments were performed with littermatecontrols and mice that were 8-12 weeks of age. The numbers of mice ineach cohort are listed in the various tables and figures.

Isoproterenol Infusion:

Alzet 2002 osmotic pumps (Durect) were sterilely loaded with 200 μLsaline or saline and isoproterenol to deliver 60 mg/kg/day for 14 days.8 week old mice were anaesthetized, and the pump was inserted sterilelysubcutaneously into the shaved back through a transverse incision madeintra-scapulae. The wound was closed with surgical staples and coveredwith betadine solution. Mice were housed separately after surgery.

Transverse Aortic Constriction:

All tools were sterilized with a Germinator 500 Dry Sterilizer andBetadine Solution (10% povidone-iodine topical solution). Anesthesia wasinduced with 5% isoflurane and maintained with 2% isoflurane and 100%oxygen at a flow rate of 1.5 L/min using a SurgiVet flow regulator vianose cone. Loss of consciousness was verified by toe pinch. Mouse furover the left chest and sternum was removed with a calcium hydroxidelotion (e.g. Nair), and the surgical site was sterilized with betadine.The skin was incised exposing the pectoralis muscle and the second leftintercostal space. The pectoralis muscle and the second rib were bluntdissected and retracted revealing the thymus within the mediastinum. Thelobes of the thymus were retracted to reveal the transverse aortic archas well as the right innominate and left common carotid arteries. Bluntdissection though the connective tissue between these two arteries andunder the aorta allowed for the passage of a 6-0 silk using a modifiedligation aid (Fine Science Tools 18062-12). A 27 gauge needle was placedon top of the aorta and the 6-0 silk was tied around the needle. Theneedle was removed, leaving a constricted aorta. The chest was closed intwo layers with 5-0 Polysorb Suture. Isoflurane administration wasterminated, and the mice were maintained on 100% oxygen by nose coneuntil conscious. Immediately post-operatively, buprenorphrine (0.05-0.1mg/kg s.c.) was administered and then q12 h prn. The mice were allowedto recover under a heat lamp until alert and active. Sham-operated micethat experience all but the placement of the aortic ligature served ascontrols.

Swimming:

8-10 week old mice were forced to swim in water tanks every day for 4weeks. The swimming tank measured >225 cm², with a depth of 15 cm and awater temperature of 30-32° C. Mice were continuously observed to avoidany drowning. The first day of training consisted of two 10-min sessionsseparated by at least 4 h. Sessions were increased by 10 min each dayuntil 90-min sessions were reached. Additional cohorts were housednormally without exercise to serve as a “sham swim” control group. Foodand water were provided ad libitum throughout the month period for allmice.

Echocardiography:

Mice minimally anesthetized with 1-2% isoflurane were studied using aVevo 770®, High-Resolution Imaging System (VisualSonics). The pressuregradient following TAC was calculated from the pulse wave Dopplervelocity at the point of ligation as follows: P=4v²; P=the inducedpressure gradient (in mmHg) and v=the velocity across the constriction(in m/s).⁷

Adult Mouse Myocytes Isolation by Langendorff Perfusion:

Mice were anesthetized using Ketamine (80-100 mg/kg) and Xylazine (5-10mg/kg) IP followed by 200 U heparin IP and cardiac excision. The heartwas placed immediately in perfusion buffer (NaCl 120 mmol/L, KCl 5.4mmol/L, Na₂HPO₄.7H₂O 1.2 mmol/L, NaHCO₃ 20.0 mmol/L, MgCl₂.6H₂O 1.6mmol/L, Taurine 5 mmol/L, Glucose 5.6 mmol/L) equilibrated with 95% O₂and 5% CO₂. The heart was attached via the aorta to the condenser outletof a Harvard Langendorff apparatus. Ca²⁺-free perfusion lasted for 5minutes with a constant rate at 2.2 mL/min at 37° C. The heart wasdigested by continuous perfusion with 25 mL buffer containing 25 mg typeII collagenase (Worthington, 315 U/mg) and 1.3 mg protease (Sigma typeXIV). After removal of the atria, the ventricles were then immersed in 5mL of the same enzyme solution for dissociation by cutting into smallpieces and by passing through a large bore pipette. The cell slurry wasfiltered through a 150-200 μm nylon mesh and the myocytes relaxed byincubation for 10 minutes in perfusion buffer containing 10 mmol/L KCl.The cells were fixed in suspension in perfusion buffer containing 3.7%formaldehyde, before morphometric analysis by light microscopy.

Histochemistry:

Heart tissue was fixed in 3.7% formaldehyde. De-paraffinized 5 μm tissuesections were stained using the Picrosirius Red Stain Kit (Polysciences)and Alexa Fluor 555 Wheat Germ Agglutinin conjugate (Invitrogen) asrecommended by the manufacturers. The cross-section area of >150myocytes in >3 distinct regions of the left ventricle were measured perheart using the wheat germ agglutinin sections. Collagen content wasassayed using the Picrosirius Red stained sections and polarized lightmicroscopy for >3 5× objective fields per heart. TUNEL staining for bothfixed cells and tissue sections was performed using the In Situ CellDeath Detection Kit, TMR red (Roche). Morphometrics and collagen contentwere measured using IPLab microscope software (BD Biosciences).

Morphometry:

Morphometric data was acquired using IPLab Software. For neonatalmyocytes, at least 6 separate images, each containing >100 cells, wereassayed for cross-section area and perinuclear prepro-ANF staining percondition for each repetition of the experiment. For adult mouse cardiacmyocytes, the maximum lengths perpendicular (width) or parallel (length)to the myofibrils were measured for >100 freshly dissociated myocytesper heart.

RNA Assays:

Total RNA was quantified with a Nanodrop 8000 Spectrophotometer (ThermoScientific) and quality controlled using with a Bioanalyzer 2100 and theRNA 6000 Nano kit (Agilent). qRT-PCR was performed using SYBR green.

The NanoString assay is based on direct, multiplexed measurement of geneexpression without amplification, utilizing fluorescent molecularbarcodes and single molecule imaging to identify and count multipletranscripts in a single reaction. For Nanostring assay, 100 ng total RNAwere hybridized in solution to a target-specific codeset overnight at65° C. The codeset contained dual, adjacently placed 50 bpoligonucleotide probes against a panel of 30 genes, one set of probesfluorescently bar-coded and the other biotinylated. The hybridizationreactions were loaded onto the NanoString Prep station which removesexcess oligonucleotides and binds the hybridized mRNA to theStreptavidin-coated cartridge surface. The cartridges were loaded ontothe NanoString Digital Analyzer, and 1155 fields of view werefluorescently scanned to count only those individual mRNAs bound to botha biotinylated and fluorescently bar-coded probe. Datasets for each RNAsample were background-subtracted and normalized using Gapdh. Invalidation assays, NanoString counts were directly proportional over 3orders of magnitude to the mRNA levels obtained by qRT-PCR and had asimilar minimum level of detection.

Statistics:

For all experiments, n refers to the number of individual mice orindividual myocyte preparations. All data are expressed as mean±s.e.m.p-values were calculated using two-tailed Student's t-tests, paired orun-paired as appropriate, and are not corrected for multiplecomparisons. Repeated symbols represent p-values of different orders ofmagnitude, for example: * p<0.05, ** p<0.005, *** p<0.0005, etc. Alldatasets involving multiple comparisons for which p-values are providedwere also significant by ANOVA, α0.05.

Discussion

RSK activity is associated with the function of the nervous system,immunity, muscle, and cancer (Anjum 2008). Human RSK2 mutations causeX-linked Coffin-Lowry syndrome, which includes mental and growthretardation and skeletal and facial anomalies, but rare cardiacabnormality. In the heart, RSK1 and RSK2 can activate the Na⁺/H⁺exchanger NHE1, and α-adrenergic-induced NHE1 phosphorylation is blockedby fmk, which inhibits all RSKs except RSK3 (Cuello 2007). The inventorsnow reveal a role for RSK3 in the cardiovascular system, regulation ofpathological myocyte hypertrophy.

Cardiac myocytes can grow in both width and length, termed concentricand eccentric hypertrophy, respectively (Kehat 2010). Concentric myocytehypertrophy involves the parallel assembly of contractile units(sarcomeres), increasing potential myocyte tension and wall thickness.In contrast, eccentric myocyte hypertrophy involves the serial assemblyof sarcomeres along the axis of contraction, mainly contributing toincreased ventricular wall area. The inventors found that RSK3 wasrequired for TAC-induced concentric hypertrophy, as well as forIso-induced myocyte growth in width in vivo. These differences can bemodeled in vitro. Interleukin-6 cytokines such as leukemia inhibitoryfactor and cardiotrophin-1 induce an elongated and eccentric phenotypefor cultured neonatal myocytes, in contrast to the symmetric growthstimulated by PE (Wollert 1996). Interestingly, the growth of thecultured myocytes tended to depend on RSK3 more when induced byα-adrenergic stimulation than by leukemia inhibitory factor (FIGS. 5 and6). The greater inhibition of PE-induced morphologic hypertrophy wasconsistent with the more robust activation of RSK by PE than leukemiainhibitory factor (FIG. 22), as well as the results obtained in vivo.

RSK3 was activated in myocytes by ERK1, ERK2, and ERK5 (FIG. 5A).Whereas RSK3 has been absent from the cardiac literature, ERK signalinghas been well-studied both in human disease and in animal models. Theautosomal-dominant human syndromes Noonan, Costello,cardiofaciocutaneous, and LEOPARD result from mutations in PTPN11, HRAS,RAF1, BRAF, MEK1, and MEK2 that activate ERK1/2 signaling (Wu 2011).These Rasopathies feature developmental delay, dysmorphic features, anddefects in multiple organ systems, often including a hypertrophicphenotype Gelb 2011). In mice, left ventricular hypertrophy has beeninduced by cardiac myocyte-specific expression of constitutively activeH-Ras and MEK1, as well as cardiac-specific deletion of the RasGTPase-activating protein neurofibromin that inhibits Ras signaling(Rose 2010, Xu 2009). Conversely, transgenic expression ofdominant-negative Raf1 inhibited the hypertrophy due to pressureoverload.

Recently, investigators have shown that cardiac myocyte-specificknockout of all 4 ERK1/2 alleles resulted in a severe, fatal dilatedcardiomyopathy without increased myocyte death (Kehat 2011). ERK1/2-nullmyocytes were longer and narrower than those from control animals.PTPN11 (Shp2) knockout that decreased ERK1/2 activation also resulted inan elongated myocyte morphology and dilated cardiomyopathy (Kontaridis2008).

Conversely, myocytes from constitutively active MEK1 transgenic micewere shorter and wider (Kehat 2011). In contrast to the ERK1/2 and Shp2knockout mice, the inventors found that deletion of the down-streameffector RSK3 resulted in a milder phenotype, with the defect inconcentric growth significant only after TAC and Iso infusion. Together,these observations are consistent with the hypothesis that ERK1/2signaling through RSK3 promotes stress-induced concentric growth ofcardiac myocytes independently of other signaling pathways that regulateeccentric hypertrophy.

The inventors found that RSK3 was activated in myocytes by not onlyERK1/2 but also ERK5. There is evidence that MEK5-ERK5 signalingprimarily induces eccentric myocyte hypertrophy (Nicol 2001), althoughERK5 also may contribute to concentric growth (Kimura 2010).

The data obtained using the RSK knockout mouse establish a function forRSK3 in pathological remodeling. Without being bound by any particulartheory, it is also possible that RSK3 has a role in physiologichypertrophy. For example, the myocytes isolated from unstressedRSK3^(−/−) mice tended to be smaller in both width and length (FIGS. 9and 29). In addition, after swimming, RSK3^(−/−) biventricular weightwas less than that of wild-type mice, albeit not significantly afternormalization by body weight (FIG. 18).

It is remarkable that even though RSK3 constitutes a minority of thetotal RSK in the myocyte (FIGS. 24 and 30), RSK3 activity is,nevertheless, required for myocyte growth. The differential anchoring ofRSK3 by scaffold proteins provides a mechanism by which RSK3 mayspecifically function in vivo. Scaffolds are likely to be most importantfor enzymes such as RSK3 that are low in abundance and that have broadintrinsic substrate specificity. RSK protein kinases catalyze thephosphorylation of RxRxx(S/T) sites and overlap in specificity withother AGC kinases (Anjum 2008). By co-localizing enzymes, their upstreamactivators, and substrate effectors, scaffolds can accelerate thekinetics of signaling, amplify responses, increase specificity in enzymecatalysis, and direct signaling to specific subcellular compartments(Good 2011). The prior art provides limited guidance with respect to RSKcompartmentation in cells or participation in multi-molecular signalingcomplexes. On mitogen stimulation, cytosolic RSK1 (and potentially otherRSK isoenzymes) can transiently translocate to the plasma membrane,whereas activated RSK tends to be enriched in the nucleus (Anjum 2008).In neurons, RSKs bind PDZ domain-containing proteins via their conservedC-terminal STxL peptides, directing the kinases to substrates involvedin synaptic transmission (Thomas 2005). By another mechanism, RSK1 bindstype 1 protein kinase A and D-AKAP-1, a mitochondrion-localized scaffold(Chaturvedi 2006, Huang 1997). Consistent with the fact that theinventors can only detect RSK3 in myocytes after immunoprecipitation,the inventors have not been able to detect endogenous RSK3 protein byimmunocytochemistry. When overexpressed at a low level, HA-RSK3 wasenriched at the nuclear envelope, the predominant location for mAKAPβ inthe cardiac myocyte (Pare 2005). By characterizing in detail theprotein-protein interaction between the unique RSK3 N terminus andmAKAPβ, the inventors have identified a new mechanism by which RSK3 canbe specifically anchored by ≧1 scaffolds that may be targeted todifferent signaling compartments.

The inventors demonstrated the functional significance of this RSK3anchoring using a competing binding peptide (mAKAP RBD) that inhibitedmyocyte hypertrophy.

The regulation of NHE1 by RSK1/2 has spurred recent interest in usingRSK inhibitors to treat heart disease (Avkiram 2008). The inventors showthat RSK3 knockout reduced TAC-induced hypertrophy without diminishingcardiac function and while inhibiting the expression of genetic markersfor pathological remodeling. RSK inhibition may have multipleapplications, including its use in acquired diseases such ashypertension (pressure overload) and for the treatment of theaforementioned Rasopathies. Recently, a Noonan syndrome mouse model(Raf1L⁶¹³V knock-in) mouse was treated with PD0325901, resulting in theattenuated progression of cardiac hypertrophy cardiomyopathy and otherNoonan characteristics (Wu 2011). Targeting of RSK3 offers analternative approach to avoid some of the harmful side effects of globalERK pathway inhibition. The use of RSK3 inhibitors that eithercompetitively bind the active site or disrupt anchoring are offered asnovel cardiac therapies.

Example 2

Remodeling of the extracellular matrix and the induction of myocardialinterstitial fibrosis is an important factor contributing to thedevelopment of heart failure in cardiac disease (Spinale 2013, Edgley2012). Increased deposition of fibrillar collagen and disruption of thenormal cellular architecture of the myocardium can result in decreasedcompliance and both diastolic and systolic dysfunction, as well asarrhythmia due to interference with the electrical conduction system.p90 ribosomal S6 kinases (RSK) are pleiotropic protein kinases that areactivated in myocytes in response to many stress-related stimuli (Anjum2008, Sadoshima 2005, Kodama 2000). The inventors have shown that type 3RSK (RSK3) is required for the induction of concentric myocytehypertrophy in mice subjected to pressure overload (Li 2013). Activatedby sequential phosphorylation by extracellular signal-regulated kinases(ERKs) and 3′-phosphoinositide-dependent kinase 1, RSK3 is one of fourRSK family members expressed in the heart (Anjum 2008). Remarkably, eventhough RSK3 comprises a minority of RSK enzyme in cardiac myocytes, RSK3is required for hypertrophy (Li 2013). Due to its role in pathologicalhypertrophy, the inventors have suggested that RSK3 targeting might bebeneficial in the prevention of heart failure. To our knowledge,however, the prior art is deficient in showing whether RSK familymembers also regulate cardiac fibrosis. In this study, the inventors nowshow a role for RSK3 in interstitial fibrosis that is independent of itsfunction in hypertrophic signal transduction.

Hypertrophic cardiomyopathy (HCM) is the most commonly inherited heartdefect (1 in 500 individuals) and the leading cause of sudden death inchildren, accounting for 36% of sudden deaths in young athletes (Maron2013). HCM is caused by dominant mutations in sarcomeric proteins thattypically induce myocyte hypertrophy and disarray and interstitialfibrosis. However, the phenotype and clinical course resulting from HCMmutations can vary such that genotype-positive patients without leftventricular hypertrophy can display myocardial fibrosis, diastolicdysfunction, and ECG abnormalities (Maron 2013). Studies usingtransgenic mice also indicate that the phenotype of HCM mutationsdepends upon genetic background (Prabhakar 2001, Michele 2002). Asdescribed below, expression of the HCM mutation Glu180Gly amino acidsubstitution of the thin filament protein a-tropomyosin (TM180) in miceof a mixed C57BL/6; FVB/N background results in a small left ventriclewith interstitial fibrosis. The inventors show that RSK3 is required inthis non-hypertrophic HCM model for the development of interstitialfibrosis and the signs of left-sided heart failure.

Methods

Supplemental Material

Reagents:

Primary antibodies included mouse 1F6 monoclonal anti-RSK3 (Abnova, cat#H00006196-M01) that detects all RSK family members (Spinale 2013), OR43rabbit anti RSK3, and N-16 goat anti-RSK3 (Santa Cruz Biotechnology).Secondary antibodies included horseradish peroxidase (HRP)-conjugateddonkey secondary antibodies (Jackson ImmunoResearch). RSK3immunoprecipitation was performed as previously described (Spinale2013).

Mice:

All experiments involving animals were approved by the InstitutionalAnimal Care and Use Committee at the University of Miami. The RSK3^(−/−)C57BL/6 mouse was mated to the TM180 transgenic FVB/N mouse (Spinale2013, Edgley 2012). All mice studied were littermates from RSK3^(−/+) XTM180; RSK3^(−/+) breedings, such that the background strain was 50:50C57BL/6; FVB/N. All four genotypes were present in typical Mendelianproportion. Unless otherwise specifies, all experiments were performedwith mice that were 16 weeks of age. Genotyping was performed at weaningby PCR using tail biopsy samples as previously described (Spinale 2013,Edgley 2012).

Echocardiography:

A Vevo 770TM High-Resolution In Vivo Imaging System (VisualSonics) witha RMVTM 707B “High Frame” Scan-head was used for imaging. Mice wereanesthetized with 1.5% isoflurane for both B-mode and M-mode imaging.

Histochemistry:

Heart tissue was fixed in 3.7% formaldehyde. De-paraffinized 5 μm tissuesections were stained using the Picrosirius Red Stain Kit (Polysciences)and Alexa Fluor 555 Wheat Germ Agglutinin conjugate (Invitrogen) asrecommended by the manufacturers. The cross-section area of >150myocytes in >3 distinct regions of the left ventricle were measured perheart using the wheat germ agglutinin sections. Collagen content wasassayed using the Picrosirius Red stained sections and linearlypolarized light microscopy for >3 4× objective fields per heart. Notethat while linearly polarized light microscopy is a highly specificassay for fibrillar collagen, the values obtained are an underestimateof total collagen content. TUNEL staining for both fixed cells andtissue sections was performed using the In Situ Cell Death DetectionKit, TMR red (Roche). Morphometrics and collagen content were measuredusing IPLab microscope software (BD Biosciences). All analyses wereperformed by a blinded investigator.

RNA Assay:

Total RNA was quantified with a Nanodrop 8000 Spectrophotometer(ThermoScientific) and quality controlled using with a Bioanalyzer 2100and the RNA 6000 Nano kit(Agilent). The NanoString assay is based ondirect, multiplexed measurement of gene expression withoutamplification, utilizing fluorescent molecular barcodes and singlemolecule imaging to identify and count multiple transcripts in a singlereaction. Briefly, 100 ng total RNA were hybridized in solution to atarget-specific codeset overnight at 65° C. The codeset contained dual,adjacently placed 50 bp oligonucleotide probes against the entire panelof genes, one set of probes fluorescently bar-coded and the otherbiotinylated. The hybridization reactions were loaded onto theNanoString Prep station which removes excess oligonucleotides and bindsthe hybridized mRNA to the Streptavidin-coated cartridge surface. Thecartridges were loaded onto the NanoString Digital Analyzer, and 1150fields of view were fluorescently scanned to count only those individualmRNAs bound to both a biotinylated and fluorescently bar-coded probe.Datasets for each RNA sample were normalized to internal positivecontrols and background-subtracted. Probe sequences are available uponrequest.

Statistics:

For all experiments, n refers to the number of individual mice. All dataare expressed as mean±s.e.m. p-values were calculated using two-tailedStudent's t-tests, paired or un-paired as appropriate, and are notcorrected for multiple comparisons. Repeated symbols represent p-valuesof different orders of magnitude: * p<0.05, ** p<0.005, *** p<0.0005.

Results

FVB/N TM180 transgenic mice were crossed with C57BL/6 RSK3 knock-outmice such that all mice were of a mixed 50:50 background. RSK3expression was slightly higher (˜25%, p=0.12) in the TM180 mice, whileabsent in RSK3^(−/−) mice, with no evidence of compensatory changes inthe expression of other RSK family members (FIG. 32). Expression of theTM180 transgene was evident by the expected change in α-tropomyosinbands detected by total protein stain (Prabhakar 2001). In these mice ofmixed lineage, the TM180 transgene induced a small heart phenotype thatincluded a reduced biventricular weight (21%) and left ventricularmyocytes with a proportionally smaller cross-section area (FIGS. 32 and34). By echocardiography, the TM180 mice had reduced left ventricularinternal dimensions, but increased contractility, i.e., both increasedfractional shortening on M-mode and increased endocardial fractionalarea shortening on B-mode (FIGS. 31 and 36). That the changes in theTM180 left ventricle were pathologically important were implied by bothan increased atrial weight and a significant, albeit small (12%)increase in wet lung weight, consistent with the presence of mildpulmonary edema and left-sided heart failure (FIGS. 32D and 34).

RSK3 knock-out had little effect on the heart in the absence of theTM180 transgene. While RSK3 knock-out did not reverse the small heartphenotype of the TM180 mouse nor prevent the atrial enlargement (FIG.32B,C and FIG. 34), the cardiac function of TM180; RSK3^(−/−) mice wasmore like wildtype mice, including a 29% lesser decrease in short axisdimension by echocardiography (FIGS. 31 and 36). Notably, the increasein fractional shortening and endocardial fractional area shortening dueto the TM180 transgene were both attenuated by ˜50% by RSK knock-out.That the more “normal” cardiac function of the TM180; RSK3^(−/−) micewas physiologically important was implied by the observation that wetlung weight was no longer increased following RSK3 knock-out (FIG. 32Dand FIG. 34).

There was no increase in cellular death associated with the TM180transgene at 16 weeks of age (˜10⁻⁴ TUNEL-positive nuclei for allcohorts, data not shown). However, trichrome staining of the TM180hearts revealed a patchy interstitial fibrosis in the myocardium notpresent in wildtype mice that was greatly reduced in the absence of RSK3(FIG. 33A,B). Likewise, picrosirius red staining showed that fibrillarcollagen content was increased by the TM180 transgene only in thepresence of RSK3 (FIG. 33C). These results were corroborated by assay ofthe expression of genes involved in cardiac function and remodeling(FIG. 35). Notably, genes involved in cardiac fibrosis, including Col8a1and Postn, (Oka 2007) encoding collagen type α1 and periostin,respectively, were induced by the TM180 transgene in a RSK3-dependentmanner (FIG. 33D,E).

Discussion

When expressed in FVB/N mice, the HCM TM180 mutation results inconcentric left ventricular hypertrophy, extensive fibrosis, atrialenlargement, and death within 5 months (Prabhakar 2001). In contrast,expression of the TM180 mutation in C57BL/6 mice resulted in noventricular hypertrophy or fibrosis and a lower heart weight (Michele2002). The inventors found that in a mixed C57BL/6; FVB/N background,the TM180 transgene induced an intermediate phenotype, includingdecreased ventricular and increased atrial weights, smaller ventricularmyocytes, interstitial fibrosis, and increased contractility byechocardiography. The TM180 mutation is thought to induce cardiomyopathyas a result of the increased Ca²⁺ sensitivity and increased maximumtension generation of TM180 filaments (Prabhakar 2001). Hence,increasing Ca²⁺ reuptake through manipulation of phospholamban and thesarco/endoplasmic reticulum Ca²⁺-ATPase 2A (SERCA2A) can ameliorate theTM180 FVB/N phenotype (Gaffin 2011). The inventors have utilized theTM180 transgenic mouse to investigate the role of RSK3 in cardiacfibrosis. While the inventors and others have found that total heartERK1/2 is activated in TM180 FVB/N mice (Gaffin 2011), in the mixedbackground mice, total ERK1/2 and RSK phosphorylation was not increased.Instead, the inventors only noted a slight increase in RSK3 proteinlevels (FIG. 32A). Importantly, RSK3 knock-out blocked the TM180associated induction of fibrotic gene expression and interstitialfibrosis, as well as improving cardiac function both in terms ofechocardiographic findings and wet lung weight. These findingscomplement our previous observation that RSK3 is specifically requiredfor pathological cardiac hypertrophy. Without being bound by aparticular theory, the inventors suggest that due to RSK3 anchoringthrough its unique N-terminal domain to scaffold proteins such as mAKAP(muscle A-kinase anchoring protein), RSK3 serves a unique function inthe heart, despite the higher level of expression of other RSKisoenzymes (Li 2013). There are reports that RSK phosphorylation ofCEBP/β is involved in pathological fibrosis of the liver and lung, butthere is no published data relating to RSK family members in the heart.The inventors suggest that specific RSK3 inhibition should now beconsidered more broadly as a therapeutic target both in hypertrophic andfibrotic heart diseases.

Example 3 mAKAP Gene Structure and the Strategy for a Conditional mAKAPAllele

The mAKAP gene contains 12 common (light blue) and 3alternatively-spliced exons (beige and yellow). A targeting vectorcontaining negative (tk) and positive (neo) selectable markers wasdesigned to conditionally delete the common Exon 9. The inventorsobtained 6 targeted ES cell clones as shown by Southern blots. Afterbreeding of targeted mice, the neo cassette was deleted by mating to aFLP recombinase transgenic. Mating to a mouse expressing cre recombinasewill result in the deletion of Exon 9 (KO allele), producing a frameshift and introduction of a stop codon (red) in Exon 10. Mousegenotyping is being performed by PCR of genomic DNA with primers 44 and45. (FIG. 37). For the western blot: mAKAP Ex9^(fl/fl);Tg(Myh6-cre/Esr1) mice (lanes 2 and 3) and mAKAP Ex9^(fl/fl) (lane 1)and Tg(Myh6-cre/Esr1) (lane 4) control mice were fed 500 mg tamoxifen/kgdry food for one week before the hearts were collected to prepare totalRNA and protein extracts. RT-PCR was performed using primers locatedwithin mAKAP exons 4 and 11 which yield a 1022 bp for wildtype (andfloxed) mRNA and a 901 bp product for a mAKAP mRNA species lacking exon9. While control b-actin mRNA was similarly detected for all samples(bottom panel), >90% less PCR product was obtained for CKO mouse hearts(top panel, lanes 2 and 3) compared to that observed with the controlhearts (lanes 1 and 4). Western blots were performed using VO54mAKAP-specific antibody. No mAKAP protein was detectable for heartextracts prepared from CKO mice (lanes 2 and 3, top panel). Equalloading was determined by Ponceau total protein stain (bottom panel).

mAKAP Ex9^(fl/fl); Tg(Myh6-cre/Esr1) mice (fl/fl; MCMTg) andTg(Myh6-cre/Esr1) (MCM Tg) at 8 weeks of age were fedtamoxifen-containing chow for one week, rested for one week and thensubjected for to 2 weeks of Transverse Aortic Constriction beforeanalysis.

Transverse Aortic Constriction:

All tools were sterilized with a Germinator 500 Dry Sterilizer andBetadine Solution (10% povidone-iodine topical solution). Anesthesia wasinduced with 5% isoflurane and maintained with 2% isoflurane and 100%oxygen at a flow rate of 1.5 L/min using a SurgiVet flow regulator vianose cone. Loss of consciousness was verified by toe pinch. Mouse furover the left chest and sternum was removed with a calcium hydroxidelotion (e.g. Nair), and the surgical site was sterilized with betadine.The skin was incised exposing the pectoralis muscle and the second leftintercostal space. The pectoralis muscle and the second rib were bluntdissected and retracted revealing the thymus within the mediastinum. Thelobes of the thymus were retracted to reveal the transverse aortic archas well as the right innominate and left common carotid arteries. Bluntdissection though the connective tissue between these two arteries andunder the aorta allowed for the passage of a 6-0 silk using a modifiedligation aid (Fine Science Tools 18062-12). A 27 gauge needle was placedon top of the aorta and the 6-0 silk was tied around the needle. Theneedle was removed, leaving a constricted aorta. The chest was closed intwo layers with 5-0 Polysorb Suture. Isoflurane administration wasterminated, and the mice were maintained on 100% oxygen by nose coneuntil conscious. Immediately post-operatively, buprenorphrine (0.05-0.1mg/kg s.c.) was administered and then q12 h prn. The mice were allowedto recover under a heat lamp until alert and active. Sham-operated micethat experience all but the placement of the aortic ligature served ascontrols.

Echocardiography:

Mice minimally anesthetized with 1-2% isoflurane were studied using aVevo 770®, High-Resolution Imaging System (VisualSonics). The pressuregradient following TAC was calculated from the pulse wave Dopplervelocity at the point of ligation as follows: P=4v²; P=the inducedpressure gradient (in mmHg) and v=the velocity across the constriction(in m/s). (FIG. 39).

Example 4 RSK3 Anchoring is Important for Neonatal Rat VentricularMyocyte Hypertrophy. FIG. 40

A. mAKAPβ complexes were immunoprecipitated using FL100 mAKAP antiserumfrom PE-treated, adenovirus-infected myocytes expressing myc-GFP ormyc-GFP-RBD (mAKAP 1694-1833) and detected with the pan-RSK 1F6 andmAKAP 211 antibodies. B. Transfected myocytes expressing GFP or GFP-RBD(green) were stained with α-actinin (blue) and ANF (red) antibodies.Bar=20 μm. C. Cross-section area of myocytes. n=5. D. Fraction ofmyocytes expressing ANF. n=3. p-values comparing to GFP-expressingsamples. t p-values comparing to no agonist control. E and F. Same as Cand D except for myocytes expressing GFP or HA-tagged RSK3 1-42.

Neonatal Rat Myocytes Isolation and Culture:

1-3 day old Sprague-Dawley rats were decapitated and the excised heartsplaced in 1×ADS Buffer (116 mmol/L NaCl, 20 mmol/L HEPES, 1 mmol/LNaH₂PO₄, 5.5 mmol/L glucose, 5.4 mmol/L KCl, 0.8 mmol/L MgSO₄, pH 7.35).The atria were carefully removed and the blood washed away. Theventricles were minced and incubated with 15 mL 1×ADS Buffer containing3.3 mg type II collagenase (Worthington, 230 U/mg) and 9 mg Pancreatin(Sigma) at 37° C. while shaking at 80 RPM. After 15 minutes, thedissociated cardiac myocytes were separated by centrifugation at 50×gfor 1 minute, resuspended in 4 mL horse serum and incubated 37° C. withoccasional agitation. The steps for enzymatic digestion and isolation ofmyocytes were repeated 10-12 times to maximize yield. The myocytes werepooled and spun down again at 50×g for 2 minutes and resuspended inMaintenance Medium (DMEM:M199, 4:1) supplemented with 10% horse serumand 5% fetal bovine serum. To remove any contaminating fibroblasts, thecells were pre-plated for 1 hour before plating on gelatin-coated tissueculture plasticware. This procedure yields >90% pure cardiac myocytes.After 1 day in culture, the media was changed to maintenance mediumcontaining 0.1 mmol/L bromodeoxyuridine to suppress fibroblast growth.

Experiments were initiated 1 day after myocyte isolation. Adenoviralinfection was performed by addition of adenovirus (multiplicity ofinfection=5-50) to the media. Plasmids and siRNA oligonucleotides weretransfected using Transfast (Promega) and Dharmafect (Thermofisher),respectively, as recommended by the manufacturers using cells culturedin maintenance medium supplemented with 4% horse serum. Starting the dayafter gene transduction, the cells were treated for as long as 2 days,as indicated for each experiment.

Immunocytochemistry:

Cultured neonatal cardiomyocytes on plastic coverslips were fixed in3.7% formaldehyde in PBS, permeabilized with 0.3% Triton X-100 in PBS,and blocked with PBS containing 0.2% BSA and 1% horse serum for 1 hour.The slides were then sequentially incubated for 1 hour with primary andAlexa fluorescent dye-conjugated specific-secondary antibodies(Invitrogen, 1:1000) diluted in blocking buffer. The slips were washedthree times with blocking buffer. 1 μg/mL Hoechst 33258 was included inthe last wash stop to label nuclei. Slides were sealed in SlowFade Goldantifade buffer (Invitrogen) for fluorescent microscopy. Wide-fieldimages were acquired using a Leica DMI 6000 Microscope.

AAV9 containing the indicated CIS plasmid encoding myc-GFP-mAKAP1694-1833 were injected into neonatal wildtype mice. 2 week TAC wasperformed and analyzed as above at 8 weeks of age. (FIGS. 41-42).

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

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1-32. (canceled)
 33. A method of treating or preventing myocardialinfarction, by administering to a patient a pharmaceutically effectiveamount of a composition that inhibits the activity of mAKAPβ.
 34. Themethod of claim 33, wherein the composition comprises a smallinterfering RNA (siRNA) that inhibits the expression of mAKAPβ.
 35. Acomposition comprising a molecule that inhibits the activity of mAKAPβ.36. The composition of claim 35, wherein the composition comprises asmall interfering RNA (siRNA) that inhibits the expression of mAKAPβ.37. The composition of claim 35, wherein the composition comprises amAKAPβ peptide.
 38. The composition of claim 37, wherein the mAKAPβpeptide comprises amino acids 1694-1833 of the mAKAPβ amino acidsequence.
 39. The composition of claim 38, wherein the mAKAPβ peptidecomprises amino acids 1735-1833 of the mAKAPβ amino acid sequence. 40.The composition of claim 35, wherein the molecule is expressed using aviral vector.
 41. The composition of claim 40, wherein the vector isadeno-associated virus (AAV).